JP6057046B2 - Method for producing microbial materials that improve soil and water pollution, suppress greenhouse gas emissions, and improve plant functionality - Google Patents

Description

The present invention is obtained by fermenting a fermentation raw material containing a plant raw material and an animal raw material using a group of microorganisms containing thermophilic microorganisms of a plurality of biological species, improving soil and water pollution, and warming while gassing inhibiting the functionality of the plant increase, especially biological defense-related genes and the expression of the high-temperature failure gene, a process for producing increased contributing microorganism material antioxidants.

  In recent years, environmental problems have become more serious on a global scale. For example, when considering agriculture from the environmental perspective, chemical fertilizers and immature compost are applied to farmland, and then contamination by nitrate ions that permeate groundwater and soil-derived warming gases. The generation of some nitrous oxide is regarded as a problem. The impact of global warming has become a cause of high temperature damage in crop cultivation, and is also an environmental factor that induces the occurrence of diseases. Furthermore, in the context of global food problems, there is a need for technologies to efficiently produce crops, and efficient operation using chemical fertilizers and pesticides is indispensable. Materials are the cause of the above environmental destruction. On the other hand, environmentally and health-friendly agricultural production techniques are required, and various technological developments such as devices and purification techniques in materials and facilities are being promoted (see Patent Documents 1 to 3). Patent Document 1 relates to a foliar spraying agent that solves the above problems by coexisting a sugar-fermented organic acid aqueous solution, magnesium or calcium, and possibly urea. In this patent document 1, although the nitrate reducing ability has been confirmed, the mechanism is also unclear, and there is no influence on other environment and plant functionality. Patent Document 2 is characterized in that the UV light source has a peak in the wavelength region of 280 to 380 nm and near the wavelength of 312 nm. This patent document 2 is the knowledge regarding the optimal illumination control applicable only in an artificial light type plant factory, and does not have the influence which acts on the functionality of other plants. Patent Document 3 relates to a method for reducing nitrate nitrogen and volatile organic compounds using alcohols. This Patent Document 3 relates to a technique for reducing nitric acid and volatile organic compounds from soil, and is not applicable to agricultural sites because it uses alcohol or the like.

  In addition, the molecular mechanisms that plants can cope with even under adverse environmental conditions have been clarified (see Non-Patent Documents 1 to 5), but technologies utilizing such mechanisms are not easily accepted by society like genetic recombination technologies. Techniques (Non-Patent Documents 6 to 8) can be found. Non-Patent Documents 1 to 5 are findings relating to HSP involvement related to disease resistance and high-temperature damage resistance, and as in the present invention, the overall effect evaluation on plants is not recognized. Further, Non-Patent Documents 6 to 8 are knowledge relating to gene recombination technology, and as in the present invention, various aspects such as nitric acid reduction, increase of antioxidants, biological defense function, high-temperature damage resistance, etc. without genetic recombination. It cannot be said that the technology has a typical function.

  On the other hand, the inventors have developed a fermentation material using a microorganism group containing thermophilic microorganisms of a plurality of species such as Bacillus brevis, Bacillus stearothermophilus, Thermopholicactinomycetes, and related species (patents). References 4-7).

JP 2006-036684 A JP 2008-086272 A JP 2002-370085 A Japanese Patent No. 3146305 Japanese Patent No. 3314302 Japanese Patent No. 3385402 International Publication WO2011 / 099514

Jarosz1 DF and Susan Lindquist1 S. (2010) Hsp90 and Environmental StressTransform the Adaptive Value of Natural Genetic Variation. Science 330: 1820-1824. Yule Liu et al. (2004) Molecular Chaperone Hsp90Associates with Resistance Protein N and Its Signaling Proteins SGT1 and Rar1to Modulate an Innate Immune Response in Plants. J. Biol. Chem. 279: 2101-2108. Jae-HeungK et al. (2000) Upregulation of an Arabidopsis RING-H2 gene, XERICO, confersdrought tolerance through increased abscisic acidbiosynthesis. The Plant Journal 47: 343-355. SnymanM and Cronje MJ. (2008) Modulation of heat shock factors accompanies salicylicacid-mediated potentiation of Hsp70 in tomato seedlings.Journal of Experimental Botany 59: 2125-2132. William B. Gurley (2000) HSP101: A KeyComponent for the Acquisition of Thermotolerance in Plants. The PlantCell, Vol. 12, 457-460. Enikeev AG et al. (2010) Tobacco cell cultures transformed by the hsp 101 gene exhibit an increasedresistance to potassium fluoride Dokl Biol Sci 430: 29-30. Montero-Barrientos M et al. (2010) Transgenic expression of the Trichoderma harzianum hsp70 gene increases Arabidopsis resistance to heat andother abiotic stresses. J Plant Physiol 167: 659-65. Prieto-DapenaP et al. (2006) Improved resistance to controlled deterioration in transgenicseeds.Plant Physiol 142: 1102-12.

  In agriculture so far, chemical fertilizers have been overused, resulting in the generation of nitric oxide polluted by nitric oxide that has penetrated into groundwater and warming gas derived from mold grown on farmland. In addition, with global warming, crop high temperature damage and disease induction have become problems. Therefore, a technology that is environmentally friendly and improves the quality of crops is needed.

The present invention is to provide a method for producing a functional microbial material that solves the above-described problems. The soil and water environment always have factors that fluctuate. Therefore, it is necessary for such factors to be multifunctionally effective not by a single bacterial species but by a complex group of microorganisms. For example, taking a soil for growing crops as an example, the moisture content of the soil is high in the rainy season, and the soil is dry in the rainy season. In response to these fluctuations, a complex response is made so that the plant function is stably improved.

I. Effects of thermophilic bacterial fermentation products on soil, groundwater contamination and greenhouse gas production 1. Reduce nitrate ion concentration in the soil. The reaction is inhibited by microorganisms (and their enzymes) that are sensitive to antibiotics such as chloramphenicol.
3. Promote denitrification from soil. In particular, it suppresses N2O production and promotes N2 production. Since N2O gas has a global warming potential approximately 300 times that of CO2, it is important to suppress its generation. Although this reaction is thought to be promoted by mold-derived P450nor, the fermented product of the present invention (containing microorganism NP-1 strain) has a function of inhibiting mold growth and gives priority to N2 gas. Genes that denitrify automatically function.
4. By the above reaction, water pollution due to nitric acid penetrating from the soil into the groundwater will be reduced as a result.
5. With salt-tolerant and alkali-resistant bacteria (genus Oceanobacillus, Virgibacillus), it is possible to purify contaminated water with a high salt concentration (around 10%).

II Effects of thermophilic bacterium fermentation products on plant functionality 1. By the action of thermophiles, the nitrate transporter in the roots is activated and nitric acid in the soil is efficiently used.
2. Develop root hairs and promote growth by auxin activation, etc. 3. Through the above reaction, it is possible to increase production with a small amount of nitrogen by efficiently using nitrogen derived from soil.
4. Grow heat-resistant plants by enzyme repair function by HSP group.
5. Increases glutathione transferase, vitamins A, C, E, etc. to produce crops rich in antioxidants. 6. Suppresses the generation of plant pathogens and plant pests due to activation of LTP and protease inhibitors.

The microbial material obtained by the production method of the present invention is composed of about 70% to about 80% by weight of plant raw material and about 30% to about 20% by weight of animal raw material. It's okay.

Moreover, the microbial material obtained by the manufacturing method of this invention may be obtained by fermenting using the microorganism group containing the microorganism of acceptance number: NITE BP-1051. Its functionality can be stabilized by microorganisms with the accession number ATCC PTA-1773.

Furthermore, the microbial group contained in the microbial material obtained by the production method of the present invention may be 108 / g to about 109 / g.

Further, the plant raw materials used for the microbial material obtained by the production method of the present invention include rice bran, wheat straw, bran , soybean meal, okara, sake lees, shochu, tea bowl, coffee lees, fruit pomace, and vegetables It may be one or more selected from the group consisting of squeezes. The animal raw material used in the present invention may be one or more selected from the group consisting of crustaceans, fish, crustacean processing residues, and fish processing residues.

Another microbial material obtained by the production method of the present invention is a functional material containing about 1 wt% to about 5 wt% of the above microbial material.

Furthermore, the present invention uses a microbial group whose accession number is ATCC PTA-1773 for the agitation step of obtaining a fermentation raw material by stirring a plant raw material and an animal raw material, and the fermentation raw material obtained in the agitation step This is a method for producing a microbial material including a fermentation process for fermentation.

According to the present invention, as shown in FIG. 1, the enzyme group having high stability of the effective microbial group in the microbial material obtained by the production method of the present invention allows nitrogen gas to permeate the nitrate ion in the soil underground. As a result of denitrification, the stimulation of roots is utilized, and the root transporter in the roots is activated. As a result, the plant grows even with a small amount of nutrient components, and the concentration of nitric acid in the plant body decreases, and at the same time, the amount of components having high antioxidant activity increases. Next, the effective microorganisms themselves in the microorganism material obtained by the production method of the present invention, and the effective microorganism group in the soil activated by them, in the form of symbiosis with the soil and the plant, the nitrogen gas in the air As a result, the concentration of glutamic acid or arginine in the plant increases. Furthermore, the expression level of the biodefense-related genes and the stress-resistant genes in the plant itself increases due to the influence of cell wall components of highly stable microorganisms in the microbial material obtained by the production method of the present invention . At the same time, the increase of highly pathogenic filamentous fungi is suppressed by the influence of cyclic lipopeptides and thermostable enzymes secreted by microorganisms in the microorganism material obtained by the production method of the present invention , and overall the quality and function of the plant Will improve. As a result of this mechanism of action, 1) Soil, groundwater contamination, water pollution, and the effects on production of greenhouse gases include the suppression of nitric acid pollution in soil and water quality and the generation of warming N2O gas released into the atmosphere. It becomes possible. Further, in industrial wastewater, wastewater treatment in a high salt concentration and high alkaline environment, which has been difficult to purify, can be performed by utilizing the characteristics of salt-resistant and alkali-resistant bacteria. 2) As an effect of thermophilic bacteria fermentation products on plant functionality, it is possible to increase production of low nitrating function and high concentration of antioxidants. At the same time, it is possible to activate a biological defense system against environmental stresses such as avoiding high temperature damage by HSP that enhances the enzyme repair function in crops and increasing repellency against pests by effects such as LTP and protease inhibitors.

It is the schematic diagram which showed the influence which acts on the soil, a plant, and the environment by the microbial material obtained by the manufacturing method of this invention . It is the pie chart which showed the bacteria gate of the complex microbial system at the time of stabilizing the microbial material obtained by the manufacturing method of this invention . It is a graph which shows the nitric acid reduction effect in a plant body depending on the addition amount of the microbial material obtained by the manufacturing method of this invention . Shows the change in the nitric acid concentration in the body of the model is a plant Arabidopsis j shepherd's purse. It is a graph which shows the nitric acid reduction effect in the soil dependent on the addition amount of the microbial material obtained by the manufacturing method of this invention , and has shown the change of the nitric acid concentration in soil. It is a graph of the experimental result which showed the length of the root of Komatsuna cultivated with the microbial material obtained by the manufacturing method of the present invention . Is a graph of experimental results showing the expression level of nitrate transporters in roots of Arabidopsis Nu Arabidopsis cultivated by microbial material obtained by the production method of the present invention. It is a graph of the experimental result which shows the change of the nitric acid concentration in the soil at the time of implementing acetylene block method with the microbial material obtained by the manufacturing method of this invention . It is a graph of the experimental result which shows the density | concentration produced from the soil at the time of implementing an acetylene block method with the microbial material obtained by the manufacturing method of this invention, and accumulate | stored as N2O. By microbial material obtained by the production method of the present invention, it is a graph of experimental results showing the concentration of accumulated ammonium ions in the body of the Arabidopsis j shepherd's purse. By microbial material obtained by the production method of the present invention, it is a graph of experimental results showing the concentration of the accumulated glutamic acid and glutamine in the body of the Arabidopsis j shepherd's purse. By microbial material obtained by the production method of the present invention, it is a graph of experimental results showing the concentration of proline accumulated in the body of the Arabidopsis j shepherd's purse.

  Next, modes for carrying out the present invention will be described, but the present invention is not limited to these embodiments.

The present invention is obtained by fermenting a fermentation raw material containing a plant raw material and an animal raw material using a group of microorganisms containing thermophilic microorganisms of a plurality of biological species, improving soil and water pollution, and warming while gassing suppressed, to provide a manufacturing how the microbial materials that have a contributing function to improve the functionality of the plant.

Microorganisms contained in the microbial material obtained by the manufacturing method of the present invention, and its metabolite acts on the microflora of the soil was fertilized with the microbial material, with adjusting the nitrate reduction reaction in the soil, the denitrification By adjusting, it is considered to regulate nitrogen circulation to soil, groundwater, plants, and atmosphere. Moreover, it is considered that the gene expression pattern of plants changes due to the change of soil microflora and induces heat shock protein (HSP) and antioxidant components.

  The microorganism group used in the present invention includes a thermophilic microorganism of a plurality of species. Specific biological species include Bacillus brevis, Bacillus stearothermophilus, Bacillus thermoamylovorans, Thermopholic actinomycetes, and closely related species thereof. Especially, it is preferable that the microorganism group used by this invention contains the microorganisms which are accession number: ATCC PTA-1773 and the microorganisms which are BP-1051.

  Accession number: ATCC PTA-1773 microorganisms are thermophilic bacteria C-1, a closely related species of Bacillus brevis, thermophilic bacteria C-3, a closely related species of Bacillus brevis, and closely related to Bacillus stearothermophilus. The thermophilic bacterium CH-4, the thermophilic actinomycete MH-1, the thermophilic or thermostable lactic acid bacterium LM-1 that is closely related to Bacillus coagulans, and the thermophilic or thermophilic bacterium that is closely related to Bacillus coagulans It is a mixed bacterium containing the thermostable lactic acid bacterium LM-2.

Microorganism material obtained by the production method of the present invention preferably contains microorganisms of about 108 cells / g to about 109 cells / g. In addition, the microorganism material obtained by the production method of the present invention has a deposit number of about 108 / g to about 109 / g: microorganisms of ATCC PTA-1773, and a deposit of about 106 / g to about 107 / g. More preferably, it contains the microorganism of number: NITE BP-863.

  The microbial material used in the present invention preferably contains a microorganism having a deposit number of NITE BP-1051 of 10% by weight to 1% by weight. Examples of the bacterial group include thermophilic microorganisms of the genera Bacillus, Lysinibacillus, Virgibacillus, Anoxybacillus, and Paenibacillus. Furthermore, microbial materials are stabilized by coexisting with Thermophiles inoculum MIROKU M2K including Meinoothermus genus, Vulcanithermus genus, Thermus genus, Oceanobacillus genus and the like of the Deinococcus-Thermus genus. These microbial groups, Thermophiles inoculum MIROKU M2K, have been preserved in Sanriku Co., Ltd. (Kitsuki City, Oita Prefecture) because they were rejected by the National Institute for Product Evaluation Technology due to complex bacteria and difficulty in culturing. In addition, as such a group of microorganisms that can coexist, the accession number PTA-1773 entrusted to ATCC can also be used.

  The plant raw material used in the present invention refers to a raw material derived from plants such as vegetables and grains, and inexpensive raw materials such as food residues can be used. Specific examples include rice bran, wheat straw, sufuma, soybean koji, okara, sake lees, shochu, tea lees, coffee lees, fruit pomace, and vegetable pomace.

  The animal raw material used in the present invention refers to a raw material derived from animals such as fish and crustaceans. Specific examples include crustaceans, fish, and processing residues thereof.

  As crustaceans, organisms called shrimps, crabs, hermit crabs and the like can be used. In addition, as fish, bottom fish pulled up by a bottom net, unused fish obtained by fishing but not sold in the market, and the like can be used. Furthermore, crustacean and fish residues processed for food can also be used.

  The fermentation raw material used in the present invention is preferably composed of about 50% by weight to about 90% by weight of vegetable raw material and about 50% by weight to about 10% by weight of animal raw material. More preferably, it is comprised of about 80% by weight plant material and about 30% to about 20% animal material.

The microbial material obtained by the production method of the present invention can improve the functionality of plants while improving soil and water pollution and suppressing the generation of greenhouse gases.

Furthermore, how to manufacture the above-mentioned microbial material of this invention, (a) stirring step, and (b) fermenting the raw material obtained in the stirring step to obtain stirring to the fermentation material and a vegetable raw material and the animal feedstock Fermenting process using a microorganism group whose accession number is NITE BP-1051.

  The stirring step (a) of the present invention is a step of stirring and mixing the above-described plant raw material and the above-described animal raw material to obtain a fermentation raw material in which each raw material is dispersed substantially uniformly. If the fermentation raw material is not sufficiently dispersed, the fermentation in the subsequent fermentation process may be incomplete. Moreover, it is preferable to grind | veget a vegetable raw material or animal raw material before stirring. This is because stirring is facilitated by pulverizing the raw material.

  The (b) fermentation process of this invention is a process of fermenting the fermentation raw material obtained at the (a) stirring process. For fermentation, a microorganism group having an accession number of NITE BP-1051 is used. The fermentation temperature is preferably about 20 ° C to about 90 ° C, more preferably about 30 ° C to about 50 ° C. The fermentation time is preferably about 5 hours to about 24 hours, more preferably about 10 hours to about 14 hours.

  Moreover, it is good to perform a (b) fermentation process with at least 2 or more several fermenters. When a plurality of fermenters are used, it is preferable to change the fermentation temperature at each stage. This is because, at each stage, fermentation with microorganisms having preference for each temperature is performed, so that functional microbial materials produced by a complex fermentation reaction can be obtained.

  Table 1 shows the closely related bacterial species of BP-1051 deposited internationally with NITE and their sequence registration.

  Table 2 shows a genus name list.

  Furthermore, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these Examples.

1. Production of functional microbial materials The functional microbial materials of Example 1 and Example 2 were produced by the method described below.

Example 1
The fermented raw material includes about 50% by weight barley meal, about 20% by weight, about 10% by weight rice bran as a vegetable raw material, and about 20% by weight as an animal raw material obtained by bottom net fishing. We used fermented marine products obtained by fermenting marine products including crustaceans such as shrimp and crabs and bottom fish. The fermented marine product includes about 108 / g to about 109 / g of microbial groups, wherein the microbial groups are about 70% to about 90% by weight of the accession number: PTA-1773 microorganisms and about 30% to about About 10% by weight of the accession number: NITE BP-1051.

  The above plant raw materials and animal raw materials were mixed and sufficiently stirred, fermented in one stage for 14 hours at 40 ° C., and dried to obtain a functional cultured feed. The functional material includes about 108 / g to about 109 / g microorganism groups, the microorganism group having about 90% to about 99% by weight of the accession number: PTA-1773 microorganisms and about 10% to about About 1% by weight of the accession number: NITE BP-1051.

(Example 2)
Fermented raw materials include about 20% by weight waste fungus bed as plant raw materials, and about 30% by weight as crustaceans such as shrimps, crabs, bottom fish, etc. Including marine products. The waste bed comprises about 108 / g to about 109 / g of microbial groups, the microbial groups having about 70% to about 90% by weight of the accession numbers: PTA-1773 microorganisms and about 30% to about About 10% by weight of the accession number: NITE BP-1051.

  The above plant raw materials and animal raw materials were mixed and sufficiently stirred, and two-stage fermentation was performed. The first stage fermentation conditions were 50 ° C. to 60 ° C. for 4 to 5 hours, and the second stage fermentation conditions were 30 ° C. to 40 ° C. and 6 to 8 hours. After the second stage fermentation, the fermented fermentation raw material was dried to obtain a functional microbial material. The functional microbial material comprises about 108 / g to about 109 / g microbial groups, the microbial groups comprising about 70% to about 90% by weight of the accession number: PTA-1773 microorganisms and about 30% by weight ~ 10% by weight of accession number: NITE BP-1051.

2. Decomposition activity of organic matter in high salt / alkaline soil and water environment
BP-1051 and Thermophiles inoculum MIROKU M2K were cultured in a heart infusion medium or the like at a salt concentration of 10%, and bacterial species having organic matter resolution were selected.

  IP-9 contained in BP-1051 is a related species of the standard strain Virgibacillus pantothenticus, but Virgibacillus pantothenticus produces a component ectoine having salt resistance. Ectoine is known as a moisturizing ingredient. In fact, IP-9 had organic matter resolution even at a salt concentration of 10% or more. It is also possible to co-culture with related species of Oceanobacillus profundus contained in the complex flora. Although this species is also known to have a high salt concentration and a high alkali concentration and has an organic matter resolving ability, in fact, a related species of Oceanobacillus profundus was found as one of the bacteria contained in Thermophiles inoculum MIROKU M2K. However, these bacterial species cannot be continuously cultured as isolated strains at this time. In any case, it can be said that the function of these bacterial species contributes to the purification of soil having a high salt concentration and a strong alkali concentration, and the water environment.

3. Soil, as an evaluation model plant of nitrate reduction for such plants, using Arabidopsis j arabidopsis (Arabidopsis thaliana), were performed in Kureha culture soil (topsoil 5 cm) as a fertilizer soil. In short, after carrying out the vernalization treatment overnight at 4 ° C, cultivation was conducted in a temperature-controlled room (23 ° C) by adding 100 ml of water every other day for 21 days under conditions of illuminance of 10,000 lux and 24-hour light period. Carried out.

The graph of the nitric acid reduction effect in a plant (FIG. 3). Graph of nitrate reduction in soil (Figure 4). As shown in FIG. 3 and FIG. 4, it was confirmed that the nitric acid concentration in the plant body and in the soil was reduced depending on the addition concentration of the microbial material obtained by the production method of the present invention . These nitric acids were not contaminated by denitrification rather than penetrating underground as shown in FIGS. Such a tendency can be confirmed also in the hydrosphere, and the effect of reducing the concentration of nitrate ion, ammonium ion and total nitrogen in water during wastewater treatment was also confirmed.

6). Lateral root induction and expression of nitrate transporter Komatsuna was adjusted to 300g with a black / red soil ratio of 8: 2, and after adding 200ml of water, the top was covered with aluminum foil and allowed to stand in a cool and dark place for one week. I put it. Then, after moving to a room with natural light and sowing, 100 ml of water was added. Thereafter, water was added every two days. Moreover, the model is a plant Arabidopsis j Arabidopsis was performed in Kureha culture soil as fertilizer soil. As described above, after carrying out the vernalization treatment at 4 ° C overnight, a certain amount was added in a constant temperature room (23 ° C) for 21 days under conditions of illuminance of 10,000 lux and 24 hours light period. And cultivated.

  As shown in FIG. 5, in the experiment using Komatsuna, root rooting increased by 20% or more. Such a tendency was confirmed in any of leaf vegetables, root vegetables, fruit vegetables, and fruit trees.

Then as a model plant, using the Arabidopsis j shepherd's purse, it was carried out in Kureha culture soil (less than overburden 3 cm) as a fertilizer soil. In short, after carrying out the vernalization treatment at 4 ° C. for 48 hours, cultivation was carried out in a thermostatic chamber (23 ° C.) while maintaining a constant water amount for 21 days under the conditions of illuminance of 10,000 lux and 24-hour light period.

Table 3, when growing Arabidopsis j arabidopsis soil depth 5 cm, shows a group of genes highly expressed by the functional material of the present invention. Table 4, when cultivated Arabidopsis j arabidopsis soil below the depth 3 cm, shows a group of genes highly expressed by microbial material obtained by the production method of the present invention.

As a result of extracting rhizome mRNA and analyzing by RT-PCR, it was confirmed that the expression level of NRT2.1, which is one of the nitrate transporter groups, increased more than twice (FIG. 6). An increasing trend was also confirmed for NRT2.6. Furthermore, as described in Table 3, it can be said that the expression level of genes related to auxin is significantly increased. Furthermore, as described in Table 4, nodulin like
The expression of protein was also considered important. In recent years, it has been suggested that nodulin contributes to root formation by flagellin, one of the extracellular components (Planta 234: 459-476, 2011).

  Thus, in addition to rooting promotion induction, there is an effect of enhancing expression of nitrate transporter and auxin-related genes in root hairs. In addition, as shown in Table 3, it was assumed that the photoresponsiveness increased because a gene that regulates phototropism such as Photootropic-responsive NPH3 family protein was also strongly expressed. Due to such complex factors, it was considered that even a small amount of fertilizer components can efficiently absorb nutrient components in the soil, and plant growth can be promoted. Regarding the plant growth-promoting effect, not only the functions of these genes but also the closely related species of Bacillus graminis, which are difficult to cultivate, among the bacterial group contained in Thermophiles inoculum MIROKU M2K. Since Bacillus graminis is a microbial endophyte that coexists with plants, there is a possibility that the bacterial species may contribute to growth promotion.

3. As evaluation model plant generation suppression factor for the greenhouse gas nitrous oxide, using Arabidopsis j shepherd's purse, it was carried out in a fertilizer soil Kureha culture soil (topsoil 5 cm). In short, after carrying out the vernalization treatment overnight at 4 ° C, cultivation was conducted in a temperature-controlled room (23 ° C) by adding 100 ml of water every other day for 21 days under conditions of illuminance of 10,000 lux and 24-hour light period. Carried out.

As shown in FIG. 6, even under acetylene atmosphere, the soil was added microbial material are obtained found by the manufacturing method of the present invention, the nitrate concentration in soil is depleted, the non-additive soil, difficult to decrease It was. Since acetylene inhibits the reaction from N2O to N2, it is possible to detect N2O by gas chromatography using the inhibition reaction. Therefore, in the soil that generates N2O, N2O can be detected even in an atmosphere that does not contain acetylene, but in the soil that generates N2, N2O can be detected only in an atmosphere containing acetylene. it can. As shown in FIG. 7, the soil was added microbial material that obtained by the production method of the present invention, it failed to detect N2O in soil conditions that are not acetylene block. In addition, N2O was detected in the acetylene-blocked soil, and it was found that N2 was preferentially released. From this, it can be said that nitrate ions in the soil are denitrified as N2 gas.

  To search for genes that promote these reactions, Throback IN et al. (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol. Ecol. 49: PCR was detected with reference to the primer sequences described in 401-417. The NirK primer used 5'-GGCGGCGCGCCGCCCGCCCCGCCCCCGTCGCCCGCCTCGATCAGATTGTGGTT-3 'as a forward primer and 5'-ATCATGGTCCTGCCGCG-3' as a reverse primer. The NirS primer used 5'-GGCGGCGCGCCGCCCGCCCCGCCCCCGTCGCCCGACTTCGGATGCGTCTTGA-3 'as the forward primer and 5'-GTCAACGTCAAGGAAACCGG-3' as the reverse primer. Furthermore, the NosZ primer used 5'-TGGGGNGAYNTBCAYCA-3 'as the forward primer, and 5'-GARCARAAGTTIGTRCARTA-3' as the reverse primer. References include Scala DJ and Kerkhof LJ (1998) Nitrous oxide reductase (nosZ) gene-specific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments.FEMS Microbiology Letters 162: 61-68, and Jones, CM, Welsh, A., Throback, IN, Dorsch, P., Bakken LR, Hallin, S. (2011) Phenotypic and genotypic heterogeneity among closely related soil-borne N2- and N2O-producing Bacillus isolates harboring the nosZ gene.FEMS Microbiol. Ecol. 76: 541-552 was used. As a result of PCR, there was a possibility that a gene group closely related to the denitrifying enzyme gene sequence derived from the genera Bradyhizobium, Herbaspirillum, and Mesorhizobium.

Table 5 shows the homology between the denitrification gene group contained in the microbial material obtained by the production method of the present invention and the denitrification gene group in the standard strain.

4). As evaluation model plant related nitrogen fixation, using Arabidopsis j shepherd's purse, it was carried out in a fertilizer soil Kureha culture soil (topsoil 5 cm). In short, after carrying out the vernalization treatment overnight at 4 ° C, cultivation was conducted in a temperature-controlled room (23 ° C) by adding 100 ml of water every other day for 21 days under conditions of illuminance of 10,000 lux and 24-hour light period. Carried out. The drying of the culture soil (reduction of water content) was adjusted by extending the water addition period for several days.

As shown in FIG. 8, an ammonium ion concentration in the body of Arabidopsis j shepherd's purse, it has been increasing tendency. Since the nitrate concentration in the plant body was extremely low and the activity of nitrate reductase was low, the source of ammonium ions was another factor and was expected to be derived from microorganisms. In addition, it is known that nitrogen fixation by microorganisms is catalyzed by a nitrogenase complex, and factors of the complex include dinitrogenase and dinitrogenase reductase. Furthermore, dinitrogenase reductase is reduced by an electron donor such as ferredoxin or flavodoxin to form ammonium ions.

Then, the cause was searched for using the primer which can amplify nifH which is a gene of dinitrogenase reductase. nifH primers include Widmer F, Shaffer BT, Porteous LA and Seidler RJ (1999) Analysis of nifH Gene Pool Complexity in Soil
and Litter at a Douglas Fir Forest Site in the Oregon Cascade Mountain Range.Appl Environ Microbiol 65: 374-380, and Poly, F., Monrozier, LJ, Bally, R. (2001) Improvement in the RFLP procedure for studying the diversity of nifH genes
Microbiol. 152: 95-103 etc. as a reference, check using 5'-TGCGACCCGAAAGCCGACTC-3 'as a reverse primer, 5'- ATGGCCATCATCTCACCGGA -3' as a reverse primer, in reference to in communities of nitrogen fixers in soil. It was decided to.

As for the bacteria in the microbial material obtained by the production method of the present invention, a bacterial species capable of growing was isolated in a nitrogen-free medium containing succinic acid serving as an electron donor. As a result, IP-23 contained in NITE BP-1051, and Lysinibacillus genus and Peanibacillus genus closely related to IP-60,75 were included. Furthermore, as shown in Tables 6 and 7, the 16S rDNA sequence was judged from the BLAST analysis results, and the Bacillus sp. 36W strain, a closely related species of Bacillus pumilus and Bacillus safensis, and the closely related Brevibacillus choshinensis and B. brevis. Brevibacillus sp. 123 strains were included, and the latter could be checked with the above-described primers, but some could not be checked with this primer. In any case, since the nucleotide sequences were not completely matched, it could be said to be a new bacterial species or subspecies. Moreover, it was suggested that the nitrogen fixation ability is demonstrated synergistically as a form of the complex bacteria group. NP-1051 contains a related species of Bacillus badius, but it is known that Bacillus badius contains a group of genes involved in nitrogen metabolism. Expected to play. In addition, as described in Table 3, in the plant grown in the soil to which the microbial material obtained by the production method of the present invention was added, the expression level of Senescence-associated protein (SEN1) is increased. Reported to be a necessary factor for symbiotic nitrogen-fixing bacteria (Plant Cell Physiol 2011 in press, http://pcp.oxfordjournals.org/content/early/2011/11/28/pcp.pcr167 .long) is interesting.

Table 6 shows the 16S rDNA sequence of the nitrogen-fixing bacterium Bacillus sp 36W strain contained in the microbial material obtained by the production method of the present invention . Table 7 shows the sequence of 16SrDNA of acquired et al are included in the microbial material, nitrogen fixing bacteria Brevibacillus sp 123 strain by the method of the present invention.

7). As evaluation model plant regulatory function of amino acid concentration, using the Arabidopsis j shepherd's purse, it was carried out in a fertilizer soil Kureha culture soil (topsoil 5 cm). In short, after carrying out the vernalization treatment overnight at 4 ° C, cultivation was conducted in a temperature-controlled room (23 ° C) by adding 100 ml of water every other day for 21 days under conditions of illuminance of 10,000 lux and 24-hour light period. Carried out. The drying of the culture soil (reduction of water content) was adjusted by extending the water addition period for several days.

As described in FIG. 10, glutamic acid in the body of the Arabidopsis j shepherd's purse, and the concentration of glutamine was significantly increased in group with addition of material. It was confirmed that arginine decomposed to glutamic acid also increased significantly. Since glutamic acid is produced from ammonium ions, it was also found that the data in FIG. 9 is consistent. In general, plant-derived glutamic acid is not only involved in the umami taste of crops, but is also an extremely important amino acid such as the expression of all antioxidant enzymes, and the microbial material obtained by the production method of the present invention improves plant quality and functionality. It could be said that it contributed. In addition, nodulin, which is one of the strongly expressed genes in Table 4, is also known to contribute to the glutamine synthesis function (Planta 234: 459-476, 2011) and is consistent with the examples according to the present invention.

Next, when the moisture content of the cultivated soil was reduced by 10%, as shown in FIG. 11, it was confirmed that proline significantly increased in the material addition group. Proline in plants is produced from glutamic acid by a reaction such as P5C reductase (Pyrroline-5-carboxylate reductase) during drying, and converted to glutamic acid by a reaction such as Proline oxidase, but under conditions with high water content Since the expression of proline oxidase more than doubled, there was no contradiction in relation to the concentration of glutamic acid. In general, proline is known to be an amino acid involved in drought tolerance and moisture retention. Therefore, the microbial material obtained by the production method of the present invention can also improve the functionality of plants. It could be said that it contributed.

  8). Expression of heat-resistant disorder genes and disease-resistant disease response genes
  Since the growth environment of plants fluctuates in nature, the model plant Shiroi under different cultivation conditions NuNazuna was cultivated and the pattern of the expressed gene was analyzed. Shiroi as a model plantNuUsing Nazuna, Kureha soil was used as a fertilizer soil. Table 3 shows data carried out in 5 cm of topsoil, and after carrying out the vernalization treatment at 4 ° C. overnight, in a thermostatic chamber (23 ° C.), the illuminance is 10,000 lux, the condition is 24 hours light period, 21 days, 100 ml. Cultivation was carried out by adding water every other day. On the other hand, Table 4 shows data carried out with less than 3 cm of topsoil, and after carrying out the vernalization treatment at 4 ° C. overnight, the temperature was controlled in a constant temperature room (23 ° C.) under the conditions of illuminance 10,000 lux and 24-hour light period. A certain amount was added and cultivated every day so as not to cut moisture. Under such conditions, DNA microarray (the same method as the previous PCT application) was performed, and a gene group strongly expressed in a model plant was comprehensively screened.

Genes expressed universally strong white Nu arabidopsis grown in soil supplemented with microbial material obtained by the production method of the present invention showed Table 3, and Table 4. In particular, the expression of heat shock protein (HSP), a molecular chaperone, was confirmed. As HSP, the expression level of HSP70 family, HSP90family, HSP101family, etc. increased.

  Among the genes whose expression levels change, regarding HSP, any HSP is a factor contributing to the resistance function against high temperature stress and various environmental stresses (Trends Plant Sci 9:, 244-252, 2004; Science 330: 1820- 1824, 2010; Plant Cell, Vol. 12, 457-460, 2000). Furthermore, RING-H2 gene, XERICO is involved in the control of the production of abscisic acid, an important plant hormone that works to control plant growth, drought tolerance, and salt tolerance (Plant Journal 47: 343-355, 2006). ).

Incidentally, the drying resistance, and it is grown in soil supplemented with microbial materials obtained by the production method of the present invention as shown in FIG. 11, when migrating soil moisture content under the conditions reduced by 10%, proline It is interesting that the degree of increase in concentration changes.

  Furthermore, as factors involved in biological defense, HSP70 and HSP90 as HSP are known to contribute to disease resistance function together with SGT1 and RAR1 (Plant Cell 19: 4061-4076, 2007; J Biol Chem 279: 2101-2108, 2004; EMBO J 27: 2789-2798, 2008). In addition, nodulin is also known to be a gene involved in disease resistance (Olivares JE et al. (2011) Nodulin 41, a novel late nodulin of common bean with peptidase activity. BMC Plant Biol 10: 134). Furthermore, the expression of Senescence-associated protein (SEN1) is regulated by salicylic acid and jasmonic acid signaling, and is assumed to be a marker gene for disease resistance. Salicylic acid is a factor involved in resistance to various pathogenic microorganisms such as viruses and bacteria, and jasmonic acid is antagonistic to salicylic acid, but is known to be a resistance inducer to environmental stress. Yes.

As a resistance gene for a pathogen, LTP (LIPID TRANSFER PROTEIN) (Nature 419: 399-403, 2002) is strongly expressed. Also trypsin and protease inhibitor / Kunitz family
Protein is known as a gene resistant to various pathogens (Molecular Plant 1: 482-495, 2008), and is an antagonist that antagonizes cell death induced by fumonisin B1, a fungus toxin. Furthermore, HPL1 (HYDROPEROXIDE LYASE 1) is a factor known to stop aphid activity (Proc Natl Acad Sci USA 98: 8139-8144,2001), and is a Cytochrome P450 71B15, which biosynthesizes phytoalexins. Putative (CYP71B15) was also strongly expressed. P450 71B15 is a cytochrome enzyme known to produce a phytoalexin called Camalexin (Plant Cell 8: 2235-2244, 1996). Camalexin is known to inhibit the growth of pathogenic filamentous fungi and bacteria. Such an enzyme is induced by environmental stress and elicitor (Plant Cell 10: 359-370, 1998), and thus functions as a soil environment change and elicitor by the microbial material obtained by the production method of the present invention. It was assumed that

In addition, Aspartyl protease (EMBO J. 2004 February 25; 23 (4): 980-988; EMBO reports 6:
282-288) may also be strongly expressed.

  In this way, expression of many biological defense-related genes is induced, but WRKY family transcription factor (Plant Mol Biol 51: 21-37, 2003) known as a gene group that can be involved in the transcriptional regulation of these genes. However, it can be said that there is no contradiction that various strong expressions are expressed. Furthermore, Glutaredoxin family protein and Glutathione S-transferase described in Table 4 are involved in antioxidant activity, and at the same time there is a tendency to increase vitamin C and vitamin E. It could be said that it improved.

  The PTA-1773 and Thermophiles-inoculum M2K used for stabilization of the material of the present invention (deposited deposit) contain chitin-degrading enzymes and lipopeptides with high antifungal activity. When applied to the soil, the abundance ratio of filamentous fungi in the soil, the plant body, or the hydrosphere can be reduced primarily.

Therefore, when the microbial material obtained by the production method of the present invention is used in the agricultural field, the biological defense genes in the plant body are activated after reducing the abundance ratio of pathogenic filamentous fungi as the growth environment outside the plant body. Therefore, it is possible to control the environment from both inside and outside the living body.

  ATCC PTA-1773

  NITE BP-1051

Claims (1)

It contributes to the growth of plants by reducing nitrate concentration in soil or water environment and plant, and promoting expression activity of nitrate transporter, promoting denitrification and fixing nitrogen, glutamine in plants, or glutamic acid, or increasing concentrations of arginine, upon drying increases the concentration of proline in plants, drought tolerance, in the manufacturing method of the microbial materials that have a function of inducing the expression of the resistance function component against pests There,
Stirring the plant raw material and the fermented raw material containing the animal raw material to obtain the fermented raw material, and the fermented raw material obtained in the stirring step with 70% to 90% by weight of the accession number: PTA-1773 microorganisms and 30 wt% to 10 wt% of accession number: NITE method for producing a microbial material, characterized in that it comprises a fermentation step of fermenting using a microorganism of the BP-1051.

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