JP6024963B2 - Novel Bacillus genus nitrogen-fixing bacteria, plant growth promoter, and plant cultivation method - Google Patents

Description

  The present invention relates to a novel Bacillus genus nitrogen-fixing bacterium belonging to the genus Bacillus and having a nitrogen-fixing ability, a plant growth promoter containing the Bacillus genus nitrogen-fixing bacterium, and a paddy rice cultivation method using the Bacillus genus nitrogen-fixing bacterium.

  Nitrogen-fixing bacteria are known as bacteria that grow symbiotically loosely in the rice rhizosphere and other crop rhizospheres, and exert growth-promoting effects by supplying nitrogen nutrients to plants. Research on nitrogen-fixing bacteria has a long history. In 1971, Yoshida et al. (Non-Patent Document 1) discovered and reported nitrogen-fixing activity in the rice rhizosphere. Watanabe and Furusaka (Non-Patent Document 2) revealed that a large number of nitrogen-fixing bacteria inhabit the rice rhizosphere. Furthermore, Baldani and Dobereiner (Non-patent Document 3) and Ladha et al. (Non-patent Document 4) isolated Azospirillum lipoferum and A. brasilense from the roots of rice. On the other hand, Greenland and Watanabe measured the nitrogen balance of long-term fertilizer continuous paddy fields in Japan and the Philippines, and estimated the biological nitrogen fixation in paddy fields to be 50-75KgN / ha.

  Based on these long-standing studies, the inoculation of Azospirillum bacteria into paddy rice increased the nitrogen fixation activity in the rhizosphere of the paddy rice, while reducing the amount of chemical nitrogen fertilizer used to supply nutrient nitrogen. Techniques that achieve a degree of yield have been tested. However, Azospirillum bacteria with nitrogen-fixing ability have a very low survival rate when they are supported on microbial carriers, and even when used as growth promoters in paddy rice cultivation and other plant cultivation, they achieve the desired effects. It was difficult to do.

Yoshida et al. (1971): Soil Sci. Soc. Am. Proc. 35: 156-157.1971 Watanabe and Furusaka (1980): (‘Microbial ecology of flooded rice soils’, in Alexander, (ed.), Advances in Microbial Ecology, Vol. 4, Plenum Publishing Corporation Baldani and Dobereiner (1980): Biol Biochem 12: 433-439, 1980) Ladha et al. (1982): Can. J. Microbiol. 28: 478-485, 1982

  Therefore, in view of the above situation, the present invention is capable of supplying nitrogen nutrition to various plants including paddy rice and is used as a plant growth promoter and exhibits a high survival rate. An object of the present invention is to provide a nitrogen-fixing bacterium belonging to the genus Bacillus and to provide a plant growth promoter and a plant cultivation method using the nitrogen-fixing bacterium.

  As a result of intensive studies by the present inventors in order to achieve the above-mentioned object, as a result of intensive studies to isolate and identify microorganisms having the desired characteristics using the soil in the field of Tokyo University of Agriculture and Technology, Fuchu City, Tokyo as a separation source Thus, it was possible to isolate and identify a novel microbial strain classified as Bacillus pumilus and having nitrogen-fixing ability. The present invention has been made based on the nitrogen fixing ability of these novel microbial strains.

The present invention includes the following.
(1) A nitrogen-fixing bacterium belonging to the genus Bacillus that has nitrogen-fixing ability and is classified as Bacillus pumilus.
(2) The Bacillus genus nitrogen-fixing bacterium according to (1), which is a Bacillus pumilus TUAT1 strain identified by the accession number NITE BP-1356.

(3) A plant growth promoter comprising the Bacillus genus nitrogen-fixing bacterium according to (1) or (2) above.
(4) The plant growth promoter according to (3), wherein the Bacillus genus nitrogen-fixing bacterium is a vegetative cell and / or a spore.

(5) A method for producing a plant, wherein the Bacillus genus nitrogen-fixing bacterium according to (1) or (2) or the plant growth promoter according to (3) or (4) is allowed to act on a target plant.
(6) The method for producing a plant according to (5), wherein the Bacillus genus nitrogen-fixing bacterium or the plant growth promoter is supplied to the rhizosphere of the plant.

(7) The method for producing a plant according to (5) or (6), wherein the plant is a gramineous plant.
(8) The method for producing a plant according to (7), wherein the gramineous plant is rice and is cultivated in paddy rice.

  Since the novel Bacillus genus nitrogen-fixing bacterium according to the present invention has an excellent nitrogen-fixing ability, it has an excellent growth promoting effect on plants. Therefore, by using the Bacillus genus nitrogen-fixing bacterium according to the present invention, a plant growth promoter having an excellent growth promoting action can be provided. Moreover, by using the Bacillus genus nitrogen-fixing bacterium according to the present invention for cultivation of plants represented by paddy rice, the growth of the plant can be promoted, and the cost for plant production can be greatly reduced.

It is a characteristic view which shows the dry matter accumulation amount of each process area in a pot test.

Hereinafter, the present invention will be described in detail.
<New Bacillus genus nitrogen-fixing bacteria>
The Bacillus genus nitrogen-fixing bacterium according to the present invention is classified as Bacillus pumilus and is a microorganism having nitrogen-fixing ability. Here, the nitrogen fixing ability means the ability to convert gaseous nitrogen molecules (N ₂ ) into nitrogen compounds (ammonia, nitrate, nitrogen dioxide, etc.). Since the Bacillus nitrogen-fixing bacterium according to the present invention has a nitrogen-fixing ability, it can supply nitrogen to plants by living in the rhizosphere. At this time, the Bacillus genus nitrogen-fixing bacterium according to the present invention is a so-called single nitrogen-fixing fungus that does not necessarily coexist with a plant, and can supply nitrogen to a wide variety of plants.

The nitrogen fixing ability can be evaluated using, for example, a nitrogen stable isotope natural abundance ratio (Δ ¹⁵ N value). The δ ¹⁵ N value can be measured using a mass spectrometer connected to an element analyzer. This method increases the atomic mass of nitrogen derived from soil due to the high natural abundance ratio of ¹⁵ N in soil, while the atomic mass of nitrogen derived from air decreases due to the low natural abundance ratio of ¹⁵ N in air. Based on the principle. That is, nitrogen derived from chemical fertilizer and nitrogen supplied by nitrogen fixation by microorganisms have a lighter atomic weight than soil-derived nitrogen atoms present in the soil. Therefore, a plant body that accumulates nitrogen supplied by nitrogen fixation by chemical nitrogen fertilizer or microorganisms contains a large amount of nitrogen having a light atomic weight. On the other hand, a plant body that has absorbed nitrogen derived from soil contains a large amount of nitrogen having a high atomic weight. Therefore, after incubating the test microorganism in the rhizosphere of the plant body, the nitrogen fixing ability of the microorganism can be evaluated by measuring the δ ¹⁵ N value of the plant body.

  The present inventors have isolated a novel microbial strain having a nitrogen-fixing ability, which is classified into Bacillus pumilus from soil in the field of Tokyo University of Agriculture and Technology, Fuchu City, Tokyo, by such a technique. The present inventor named this new microbial strain as Bacillus pumilus TUAT1 and incorporated the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (NITE Patent Microorganism Deposit Center: Kazusa Kisarazu City, Chiba Prefecture 292-0818) 2-5-8 in Kamaishi) International deposit has been made on May 10, 2012 under the accession number NITE BP-1356.

  The Bacillus genus nitrogen-fixing bacterium according to the present invention is classified into the Bacillus pumilus TUAT1 strain specified by the accession number NITE BP-1356 and the same strain as the TUAT1 strain, and has a nitrogen-fixing ability. It will contain microorganisms.

  Further, the Bacillus pumilus TUAT1 strain specified by the accession number NITE BP-1356 has 16S rDNA containing the base sequence shown in SEQ ID NO: 1. Therefore, the Bacillus genus nitrogen-fixing bacterium according to the present invention contains 16S rDNA containing a base sequence having a homology of 95% or more, preferably 98% or more, more preferably 99% or more with respect to the base sequence shown in SEQ ID NO: 1. Bacillus pumilus having a nitrogen-fixing ability.

<Plant growth promoter>
By utilizing the nitrogen fixing ability of the Bacillus genus nitrogen-fixing bacterium according to the present invention, nutrient nitrogen can be supplied to the plant. That is, the Bacillus genus nitrogen-fixing bacterium according to the present invention can be used as a plant growth promoter. In addition, you may use the Bacillus genus nitrogen fixed bacteria which concern on this invention in any state of a vegetative cell and a spore.

When the vegetative cells of the Bacillus genus nitrogen-fixing bacterium according to the present invention are used as a plant growth promoter, for example, culture is performed under appropriate conditions using a medium as exemplified below. In other words, usable media include NFb medium (malic acid 5.0 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ .7H ₂ O 0.2 g, NaCl 0.1 g, CaCl ₂ 0.02 g, Na ₂ MoO ₄ .2H ₂ O 0.002 g, MnSO ₄ · H ₂ O 0.01 g, KOH 4.5 g, Fe EDTA (1.64% W / V%) 4.0 ml, Biotin 0.1 ml are dissolved in 1 l of distilled water, and the pH is adjusted to 6.8 with NaOH or KOH) , Trypticase soy medium (Nippon Becton Dickinson Co., Ltd.) and the like. Moreover, as culture conditions, the culture temperature can be 20 to 30 degrees, preferably 25 to 28 degrees, and the medium pH is desirably 6.8.

  When the spore of the Bacillus genus nitrogen-fixing bacterium according to the present invention is used as a plant growth promoter, the spore-forming medium that can be used may be the above-mentioned medium, and the agar plate and the liquid medium are kept at 25 ° C. for about 1 week. It is obtained by placing. When left in a liquid medium, the spores tend to agglomerate and can be prevented by adding a certain amount of a suitable surfactant. Inoculation in the spore state maintains the survival of the spore for several months and increases the stability of the inoculation effect.

  When the Bacillus nitrogen-fixing bacteria (vegetative cells and / or spores) according to the present invention cultured as described above are used as plant growth promoters, the Bacillus nitrogen-fixing bacteria (vegetative cells and / or spores) according to the present invention are used. May be used alone, but the Bacillus genus nitrogen-fixing bacteria and other optional components may be blended to form a specific preparation. Examples of the form of the preparation include liquids, powders, granules, emulsions, oils, suspensions, wettable powders, water solvents, granules, pastes, capsules, aerosols (aerosols) and the like. it can.

  Other optional formulations include, for example, carriers for supporting Bacillus genus nitrogen-fixing bacteria such as liquid carriers and solid carriers, surfactants (emulsifiers, dispersants, antifoaming agents, etc.), adjuvants and the like. Examples of the liquid carrier include phosphate buffer, carbonate buffer, and physiological saline. Solid carriers include natural mineral powders such as kaolin, clay, talc, bentonite, chalk, quartz, attapulgite, montmorillonite, white carbon, diatomaceous earth, synthetic mineral powders such as silicic acid, alumina, silicate, crystalline cellulose, corn starch And high molecular weight natural products such as gelatin and alginic acid. In addition, as the solid carrier, for example, inorganic substances such as vermiculite, silica sand, mica, pumice, gypsum, calcium carbonate, dolomite, magnesium, slaked lime, phosphorus lime, zeolite, and ammonium sulfate may be used. Further, as the solid carrier, for example, plant organic substances such as soybean powder, tobacco powder, walnut powder, wheat flour, wood powder, starch, crystalline cellulose and the like may be used. Furthermore, as solid carriers, there are synthetic or natural polymer compounds such as coumarone resin, petroleum resin, alkyd resin, polyvinyl chloride, polyalkylene glycol, ketone resin, ester gum, copal gum, dammar gum, carnauba wax and beeswax. Waxes and ureas may be used.

  As the emulsifier or dispersant, a surfactant is usually used. As the surfactant, nonionic, cationic, anionic and zwitterionic ones are used, but usually nonionic and / or anionic ones are preferably used. Suitable nonionic surfactants include, for example, those obtained by polymerizing and adding ethylene oxide to higher alcohols such as lauryl alcohol, stearyl alcohol and oleyl alcohol; those obtained by polymerizing and adding ethylene oxide to alkylphenols such as isooctylphenol and nonylphenol; Polymerized addition of ethylene oxide to alkyl naphthols such as butyl naphthol and octyl naphthol; Polymerized addition of ethylene oxide to higher fatty acids such as valmitic acid, stearic acid and oleic acid; Mono- or stearic acid, dilauryl phosphate, etc. Polymerized addition of ethylene oxide to dialkyl phosphoric acid; Polymerized addition of ethylene oxide to amines such as dodecylamine and stearamide; Sorbitan What higher fatty acid ester of a polyhydric alcohol and it shall was ethylene oxide polymerized addition; ethylene oxide and propylene oxide that was polymerized addition, polyhydric fatty acid esters of an alcohol, such as dioctyl succinate. Suitable anionic surfactants include, for example, alkyl sulfate salts such as sodium lauryl sulfate and oleyl alcohol sulfate ester amine salts; alkyl sulfonate salts such as sodium sulfosuccinate dioctyl ester and sodium 2-ethylhexene sulfonate. Aryl sulfonates such as sodium isopropyl naphthalene sulfonate, sodium methylene bisnaphthalene sulfonate, sodium lignin sulfonate, sodium dodecylbenzene sulfonate; phosphates such as sodium tripolyphosphate;

The content of the Bacillus genus nitrogen-fixing bacterium of the plant growth promoter according to the present invention is not particularly limited, but can be 10 ⁷ to 10 ⁸ cfu / ml.

On the other hand, the plant growth promoter configured as described above promotes the growth of plants by being supplied to the rhizosphere of various plants. The target plant is not particularly limited, and examples thereof include dicotyledonous plants, monocotyledonous plants such as Brassicaceae, Gramineae, Eggplant, Legume, Willowaceae and the like (see below), It is not limited to these plants.
Brassicaceae: Arabidopsis thaliana, Brassica rapa, Brassica napus, Brassica campestris, cabbage (Brassica oleracea var. Capitata), Chinese cabbage (Brassica rapa var. Pekinensis), Chingensai (Brassica rapa var, chin) Brassica rapa var. Rapa), Nozawana (Brassica rapa var. Lancinifolia), Komatsuna (Brassica rapa var. Peruviridis), Pakchoi (Brassica rapa var. Chinensis), Japanese radish (Raphanus sativus) (Wasabia japonica).
Eggplant family: tobacco (Nicotiana tabacum), eggplant (Solanum melongena), potato (Solaneum tuberosum), tomato (Lycopersicon lycopersicum), red pepper (Capsicum annuum), petunia (Petunia) and the like.
Legumes: Soybean (Glycine max), pea (Pisum sativum), broad bean (Vicia faba), wisteria (Wisteria floribunda), groundnut (Arachis hypogaea), Lotus corniculatus var. Japonicus, common bean (Phaseolus vulgaris), azuki bean (Phaseolus vulgaris) Vigna angularis), Acacia, etc.
Asteraceae: Chrysanthemum morifolium, sunflower (Helianthus annuus), etc.
Palms: oil palm (Elaeis guineensis, Elaeis oleifera), coconut (Cocos nucifera), date palm (Phoenix dactylifera), wax palm (Copernicia), etc.
Ursiaceae: Rhis succedanea, Cashew nutcrest (Anacardium occidentale), Urushi (Toxicodendron vernicifluum), mango (Mangifera indica), pistachio (Pistacia vera), etc.
Cucurbitaceae: Pumpkin (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo), cucumber (Cucumis sativus), crow cucumber (Trichosanthes cucumeroides), gourd (Lagenaria siceraria var. Gourda), etc.
Rosaceae: Almond (Amygdalus communis), Rose (Rosa), Strawberry (Fragaria), Sakura (Prunus), Apple (Malus pumila var. Domestica), etc.
Dianthus: Carnation (Dianthus caryophyllus).
Willow family: Poplar (Populus trichocarpa, Populus nigra, Populus tremula) etc.
Gramineae: corn (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), bamboo (Phyllostachys), sugar cane (Saccharum officinarum), napiergrass (Pennisetum pupureum), elian raven (Erian) ), Miscanthus virgatum, Sorghum switch glass (Panicum), etc.
Lily family: Tulip (Tulipa), Lily (Lilium), etc.

  Among these, the plant growth promoter according to the present invention is preferably intended for gramineous plants represented by rice (Oryza sativa). In particular, the plant growth promoter according to the present invention is effectively used in paddy rice cultivation.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, the technical scope of this invention is not limited to a following example.
[Example 1] <Isolation of Bacillus pumilus TUAT-1 strain>
It is isolated from the soil of the field of Tokyo University of Agriculture and Technology, Fuchu City, Tokyo, using an NFb-nitrogen-free anti-fluid culture medium. It has the characteristic of forming a cell mass about 5 mm below the surface of the fluid culture medium. And confirmed rooting promotion effect on rice.

  DNA extracted from microorganisms with nitrogen-fixing ability isolated as described above, 16s-rRNA gene region amplified by PCR method, DNA base sequence determined using DNA analyzer, official DNA information storage agency Classification was attempted by comparison with the nucleotide sequence. The base sequence of the 16s-rRNA gene region is shown in SEQ ID NO: 1. As a result of the Blast search in the Japan DNA Data Bank (DDBJ), the nucleotide sequence of SEQ ID NO: 1 was categorized as Bacillus pumilus.

  Bacillus pumilus isolated in the present example is known for Bacillus pumilus (for example, Bacillus pumiluis, for example, in that it has a nitrogen fixing ability, an indole acetic acid producing ability, and a strong rooting promotion particularly in rice plants, particularly paddy rice. The SYBC-W strain (laccase production strain) and the Bacillus pumilus AUCAB16 strain (strain that lives in the cocoon)) were judged to be new strains different from the known strains. The Bacillus pumilus isolated in the present example was named Bacillus pumiluis TUAT1 strain, and was incorporated by the National Institute of Technology and Evaluation Technology Patent Microorganism Depositary Center (NITE Patent Microorganism Depositary Center: 2 Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 292-0818) -5-8) was deposited internationally on May 10, 2012 as accession number NITE BP-1356.

[Example 2]
In this example, the nitrogen fixing ability and the plant growth promoting action based on the Bacillus pumiluis TUAT1 strain (Accession No. NITE BP-1356) isolated and identified in Example 1 were verified.

<Test method>
<Adjustment of test strain and inoculum>
Bacillus pumilus TUAT-1 strain was cultured with shaking in 5 L NFb liquid medium containing 1 g / L ammonium chloride for 1 week at 25 ° C., and collected by centrifugation at 4 ° C., 6000 rpm for 30 minutes. Next, sterilize 1 kg of black soil from the farm attached to Tokyo University of Agriculture and Technology, passed through a 2 mm sieve, in an autoclave at 121 ° C for 40 minutes, mix the collected cells in 100 mL of sterilized distilled water, 28 ° C, It was left to stand for 1 week under the maximum field capacity and matured. The bacterial density of the inoculum was measured by a dilution plate method and adjusted to 10 ⁷ to 10 ⁸ CFU / g.

<Inoculation method>
In the case of a pot test, the TUAT-1 strain, such as “Leaf Star”, was inoculated so that the seedling root was wrapped with 200 g of the inoculum at the time of seedling transplantation. In the field test, 100 g of the inoculum was suspended in water on a vat to form a suspension, and the seedlings were inoculated by immersing the roots of the transplanted seedling for 1 hour.

  Examination of the inoculation method revealed that in the case of rice, a stable effect can be obtained by inoculating the above inoculum at the time of sowing and then inoculating the same multiple times. In addition, stable inoculation can be established by immersing the transplanted seedlings in the inoculum with amberite.

  “Leaf Star” was bred by Okawa et al. As a cultivar that was newly produced in Nagatoro, with high biomass production and high starch content in rice straw (Biomass production and lodging resistance in 'Leaf Star', a new long-culm rice forage cultivar, Ookawa, T., K. Yasuda, M. Seto, K. Sunaga, H. Kato, M. Sakai, T. Motobayashi, S. Tojo and T. Hirasawa, Plant Production Sceince, 13 (1): 56-64 2010).

<Pot test>
1 / 5000a porcelain pot sieved to Tokyo University of Agriculture and Technology, Faculty of Agriculture, Metropolitan Field Science Education and Research Center FM Honmachi paddy field, filled with fertilizer and filled with leaf star seedlings grown for 20 days . The pot has three fertilization stages. That is, the customary zone (5kg / 10a), the reduced fertilizer zone (2kg / 10a) with 60% reduction of nitrogen, and the non-fertilized zone (0kg / 10a). Furthermore, each bacterial inoculation zone and non-inoculation zone were set up for a total of 6 treatment zones. Phosphoric acid and potassium were applied at 5kg / 10a in the conventional and reduced fertilizers, and neither was given to the non-fertilizers. The pots were placed in the glass room of Tokyo University of Agriculture and Technology at random in three treatment zones and cultivated under natural light. 106 days after the start of cultivation, the plant body was collected together with the roots and divided according to the parts of the ears, foliage and roots. After drying at 70 ° C. for 48 hours in a ventilation dryer, the dry weight was measured. As chemical analysis items, dried plants were subjected to plant nutrient analysis and 15N natural abundance measurement as described below.

<Field test>
The field inoculation tests were conducted in FM Honmachi Mizuta (Tama River alluvial soil) and Ogata Village, Akita Prefecture, Tokyo Metropolitan University of Agriculture and Technology. At the Tokyo University of Agriculture and Technology, there are three fertilization stages: the conventional zone (10kg / 10a), the reduced fertilizer zone (7kg / 10a) with 30% nitrogen reduction, and the non-fertilized zone (0kg / 10a). Each of them had a fungus inoculation zone and a non-inoculation zone, for a total of 6 treatment zones. Phosphoric acid and potassium were applied at 10kg / 10a in the conventional and reduced fertilizers, and neither was given to the non-fertilizers. Paddy rice “Leafstar” seedlings grown for 20 days were transplanted at 3 per plant so that the planting density was 22.2 strains / m ² . Survey items were 1) As growth survey items, five consecutive strains from within the community of each treatment area were selected as growth survey strains, and plant height, number of stems, and leaf color (SPAD value) were measured every week. A SPAD meter (MINOLTA, SPAD-502) was used to measure the leaf color. 2) As a growth analysis item, on the 127th day after transplantation, which is the ripening period, the above-ground part of 2 Articles 8 strains (16 strains in total) was collected from each treatment area, and the above-ground fresh weight was measured. Among them, 4 strains having an average fresh weight were classified according to the parts of ear, leaf blade (referred to as leaf), stem and leaf sheath, and dead part (referred to as stem), and dry weight was measured. 3) Also, as chemical analysis items, dry matter was subjected to plant nutrient analysis and ¹⁵ N natural abundance measurement as described below.

  In Ogata Village, the seedling box was immersed in a suspension of inoculum before machine transplantation, and then transplanted with a transplanter. Measurement items were plant height, number of stems, leaf color (SPAD), and yield components.

<Plant nutrient analysis>
In order to evaluate the absorption of nitrogen, phosphoric acid, and potassium in the plant body, each concentration and accumulation amount in the plant body were determined. First, the dried plant bodies obtained in the pot test and the field test were pulverized with a pulverizer, and the plant powder was wet-decomposed by the concentrated sulfuric acid-hydrogen peroxide method. Thereafter, the nitrogen concentration in the decomposition solution was colorimetrically determined by the indophenol method and the phosphorus concentration by the vanadomolybdic acid method, and quantified using a spectrophotometer (Shimadzu, UV-160). The potassium concentration was measured by diluting the sample solution 5 times or 10 times and using a flame photometer (Hideko Seiki, FLA type). From these, the concentration of nitrogen, phosphorus, and potassium contained in the plant body was obtained, and the accumulated amount of these elements in the plant body was calculated by multiplying by the dry weight value.

^<15 N measurement of natural abundance ratio>
In order to confirm whether the nitrogen fixed by the inoculum has transferred to the leaf star, a part of the dried plant powder obtained in the pot test and the field test was treated with an ultrafine grinder, and the natural presence of 15N The ratio was used for measurement. The measurements were requested and analyzed at the University of California Davis Stable Isotope Facility.

<Result>
<1 / 5000a pot test>
<Dry weight>
Table 1 shows the measured dry weight of each leaf star in the heading period.

  As shown in Table 1, in the root part, when the non-inoculated section of the conventional section is set to 100 in terms of the index, the inoculated section is 205, and the dry weight of the root amount has increased about twice as much by the inoculation. Also, in the reduced fertilizer group, the index of 137 was shown compared to the conventional non-inoculated group, and there was a tendency for the root dry matter weight to increase, and it was found that inoculation with TUAT1 strain promoted root growth. In the non-fertilization zone, no effect on the roots was observed, and an interaction effect was observed between the fertilization stage and the inoculation with a risk rate of 5%. It was shown that the effect is more manifested. The value of total dry weight showed a significant increase due to the amount of nitrogen applied and bacterial inoculation (P = 0.01), and a significant increase in dry matter accumulation was observed in the reduced fertilizer and conventional plots compared to the control plot. In addition, the value of total dry weight in the inoculated area with reduced fertilizer was similar to that in the non-inoculated area, suggesting that the reduced nitrogen content could be compensated by inoculation. Fig. 1 shows the correlation between fertilization stage and dry weight. As shown in FIG. 1, a high correlation was observed between the fertilization stage and the dry matter weight, and it became clear that the dry matter accumulation amount increased as a result of inoculation with the bacteria.

<Nutrient analysis>
As a result of analysis of variance, there was a significant difference in the effect of fertilization stage and fungus inoculation (P = 0.05, P = 0.01) on the amount of nitrogen accumulation in leaf star per pot. Absorption was shown to be enhanced (Table 1).

  In addition, regarding nitrogen and potassium accumulation, similar to nitrogen, significant differences were observed in the stage of fertilization and the effect of each inoculation of bacteria (Table 1), and absorption of phosphorus and potassium was promoted by fertilization and inoculation of bacteria. It was shown that.

<Origin of accumulated nitrogen based on δ ¹⁵ N value>
Table 2 shows the values of δ ¹⁵ N in the inoculation and non-inoculation sections of the root and foliage in the pot test.

As shown in Table 2, it was found that when chemical fertilizer inoculation increased, the δ ¹⁵ N value decreased in the non-inoculated group compared with the non-fertilized group, and the atomic weight of nitrogen became lighter. This indicates that chemical nitrogen fertilizer directly affects the δ ¹⁵ N value. On the other hand, in the inoculated area, the value tended to increase slightly compared to the non-inoculated area. From this result, it was found that rice inoculated with TUAT1 strain absorbed a lot of nitrogen in the soil together with chemical nitrogen fertilizer by rooting compared to non-inoculated area.

<Field test 1 (Agricultural and industrial paddy field)>
<Dry weight>
Table 3 shows the measured values of dry weight of leaf stars above the ripening stage.

  As a result of analysis of variance shown in Table 3, a significant difference was observed for the factors of fertilization and inoculation with the total dry weight of the above-ground part (P = 0.01). In the comparison at each fertilization stage, the inoculation group significantly exceeded the control group in the conventional and reduced fertilization groups, and the inoculation effect was recognized. In the non-fertilized area, the effect of inoculation was not observed. In addition, an overall interaction was observed between both the fertilization level and the inoculation factor (P = 0.05), suggesting that the inoculation effect appears as the fertilization stage increases. In terms of the above-ground parts, a significant increase in dry matter weight was observed in all areas in the conventional area and in the leaves and stems in the reduced area.

  In multiple comparisons between all 6 treatment groups, there was no significant difference between the reduced fertilizer-inoculated group and the conventional non-inoculated group. The possibility was suggested.

<Nutrient analysis>
Table 3 also shows the values of nitrogen, phosphorus and potassium accumulation in leaf stars during the ripening period. As can be seen from this value, in each nutrient, significant effects of both fertilization and fungal inoculation factors were observed in the whole aerial part and by site (P <0.05). Furthermore, the interaction between the two factors was recognized in the whole aerial part as well as the dry weight value.

  In all fertilization stages, the inoculation of the nitrogen was shown to increase by inoculation with bacteria, and the increase was 17% in the non-fertilized area, 34% in the reduced fertilizer area, and 37% in the conventional area. In the pot test, there was no effect of inoculation on the nitrogen concentration in the plant, but in the field test, there was a clear effect of inoculation on the nitrogen concentration, and the nitrogen concentration and fertilization stage were extremely high A correlation (R2> 0.99) was observed. In other words, unlike the results of pot tests, the promotion of nitrogen accumulation in plants by inoculation with bacteria in field tests is achieved by increasing the nitrogen concentration in the plants by further adding to the amount of fertilization. It was happening.

  In addition, with respect to phosphorus and potassium accumulation, a significant effect was observed by fertilizer application and inoculation in the same manner as nitrogen, and in the whole plant, significant accumulation by inoculation in the reduced fertilizer and customary areas for phosphorus and in the conventional area for potassium. An increase in quantity was observed (Table 3). The phosphorus and potassium concentrations in the plants tended to correlate with the amount of nitrogen applied, and the absorption of phosphorus and potassium was promoted with the amount of nitrogen applied.

<Field test 2 (Ogata village paddy field in Akita Prefecture)>
We verified whether TUAT1 inoculation was applicable to large-scale mechanized rice cultivation in Ogata Village, Akita Prefecture. Table 4 shows the growth situation in the farmer's field.

  As shown in Table 4, regarding plant height, there was no significant difference between non-inoculation and inoculation for paddy rice in F and T fields. Regarding the SPAD value of leaves, the T field was slightly higher than the F field, indicating that the nitrogen supply capacity in the soil of the T field was higher than that of the F field. Regarding the number of stems, in both rice fields, in the highest splitting period (July 23), the number of stems in the inoculated section exceeded the non-inoculated section, and even after the earliest sunrise on August 14, The number of stems in the inoculated group exceeded that in the non-inoculated group. Other field tests (data not shown) showed similar effects.

  In addition, the effect of the inoculation of TUAT1 strain in the farm field of Ogata Village in Akita Prefecture on the yield and yield components of the rice cultivar "Komachi" was described.

  As shown in Table 5, in the F field, there was no significant difference in the thousand grain weight and ripening rate between the TUAT1 non-inoculation and the inoculation, and the number of spikelets decreased rather. However, the number of spikes in the inoculated area increased by 22.4% compared to that in the non-inoculated area, and as a result, the yield in the inoculated area increased by 32.8%. In addition, in the T field, there was no significant difference in the thousand grain weight, ripening rate, and number of spikelets in the non-inoculated section and the inoculated section, but the number of spikes in the inoculated section increased by 14.6% compared to that in the non-inoculated section. As a result, the yield in the inoculated area increased by 17.9% compared with that in the non-inoculated area.

  Thus, it was found that the seed yield of rice increased as the number of ears increased when the TUAT1 strain was inoculated in the conventional fertilized area.

Claims (7)

A Bacillus genus nitrogen-fixing bacterium which is a Bacillus pumilus TUAT1 strain having a nitrogen-fixing ability and specified by the accession number NITE BP-1356 . A plant growth promoter having a rooting promoting effect, comprising the Bacillus genus nitrogen-fixing bacterium according to claim 1 . The plant growth promoter according to claim 2, wherein the Bacillus genus nitrogen-fixing bacteria are vegetative cells and / or spores. It reacted with claim 1, wherein the Bacillus nitrogen fixing bacteria or claim 2 or 3, wherein the plant growth promoting agent to the target plants Ru to promote rooting, method of manufacturing plants. The method for producing a plant according to claim 4, wherein the Bacillus genus nitrogen-fixing bacterium or the plant growth promoter is supplied to the rhizosphere of the plant. The method for producing a plant according to claim 4 or 5, wherein the plant is a gramineous plant. 7. The method for producing a plant according to claim 6 , wherein the gramineous plant is rice and is cultivated in paddy rice.

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