JP6296776B2 - Biofertilizer manufacturing method - Google Patents

Description

  The present invention relates to a biofertilizer that uses microbial materials, and in particular, relates to a microbial fertilizer that is excellent in preservability and has high crop fixing ability.

  Conventionally, chemical fertilizers have been used mainly for general crop production from the viewpoint of cost advantage, quick effect, and high fertilization efficiency, and have contributed to increased crop production.

  However, due to various food quality and safety issues and environmental load problems due to the leaching of fertilizer components, etc., there has been a tendency in recent years to reexamine cultivated body types that depend on chemical substances such as chemical fertilizers and agricultural chemicals. is there. In addition, there is an increasing tendency to pursue environmental conservation agriculture in response to the production of safe, safe, and tasty crops desired by consumers. Along with this, application of materials containing microorganisms tends to increase.

  In order to use materials containing microorganisms, microbial materials that have been marketed so far must be stored for a long period of time (for example, 3 months) unless they are stored under refrigerated conditions or a single fungus paste such as skim milk is prepared. ), The number of bacteria and the problem that it was difficult to maintain the effect.

  As countermeasures against such problems, it is possible to unify bacteria contained in materials (excluding miscellaneous bacteria) by adding ultraviolet rays, adding plant residues, etc., and increasing the activity of only the target bacteria It was generally done.

  In such microbial materials, some proposals have been made for the purpose of maintaining the number of bacteria and the effect thereof. For example, Patent Document 1 proposes a microorganism material that is granulated by adding a microorganism culture solution or a supernatant of a microorganism culture solution to a fertilizer or a porous material. Further, Patent Document 2 proposes that a Bacillus genus bacterium can be sporulated to maintain a high survival rate and make a feed with a high bacterial concentration.

JP 2002-363561 A JP 2000-217567 A

  An object of the present invention is to propose a biofertilizer capable of maintaining the number of bacteria and the effect for 6 months or more at room temperature (around 25 ° C.).

  The present invention is a biofertilizer having a spore ratio of 100%, which is obtained by culturing Bacillus bacteria and then heat-treating it at a temperature range of 65 ° C to 80 ° C for 60 minutes or more.

  According to the present invention, it is possible to provide a biofertilizer capable of maintaining the number of bacteria and the effect for 6 months or more at room temperature (around 25 ° C.).

  In addition, the characteristics of Bacillus bacteria such as growth promotion, which have been recognized in the past, are expressed more effectively. Especially, the root area of applied crops (for example, rice) is expanded, root colonization is improved, and roots are improved. The biofertilizer which can improve a weight increase effect and a nutrient absorption promotion effect can be provided.

The figure showing the dry matter accumulation | storage amount of each process section in the 1 / 5000a pot test using Bacillus pumilus TUAT1 strain | stump | stock (NITE BP-1356). It is the reference photograph showing the examination result which compared the influence which the difference in the vegetative type and the spore type of Bacillus pumillus TUAT1 strain (NITE BP-1356) has on the root elongation of rice, the left side is spore type, and the right side is Nutritional type.

  Conventionally, Bacillus pumilus has been known to be a useful bacterium having properties such as growth promotion, disease suppression, odor reduction, and proteolytic properties.

  The inventors of the present application select the Bacillus pumilus TUAT1 strain (NITE BP-1356) from the Bacillus pumilus, and examine the effects of promoting root absorption and nutrient absorption when applied to rice as follows. It was.

  The Bacillus pumillus TUAT1 strain has been deposited internationally at the Patent Microorganism Depositary, and the deposit number is NITE BP-1356.

(Adjustment of test strain and inoculum)
Bacillus pumilus TUAT1 strain (NITE BP-1356) (hereinafter sometimes referred to as “TUAT1 strain”) was cultured with shaking in 5 liters of NFb liquid medium containing 1 g / liter of ammonium chloride at 25 ° C. for 1 week. The cells were collected by centrifugation at 4 ° C. and 6000 rpm for 30 minutes.

Next, 1 kg of black soil from the farm attached to Tokyo University of Agriculture and Technology passed through a 2 mm sieve was sterilized in an autoclave at 121 ° C for 40 minutes, and the collected cells were mixed in 100 mL of sterilized distilled water. The mixture was allowed to stand for 1 week under 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, TUAT1 strain “Leaf Star” and the like were inoculated so that the seedling roots were 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 several times.

  In addition, stable inoculation can be established by immersing the transplanted seedling in the inoculum overnight.

(Pot test)
Fertilizer was mixed and filled in the gray lowland soil of the Field Science Research Center FM Honmachi paddy field attached to the Tokyo University of Agriculture and Technology, which was sieved into a 1 / 5000a porcelain pot, and leaf star seedlings grown on the 20th were transplanted. The pot was provided with three fertilization stages. That is, it is a customary zone (5 kg / 10a), a reduced fertilizer zone (2 kg / 10a) with 60% reduction in nitrogen, and a non-fertilized zone (0 kg / 10a).

Furthermore, each inoculation zone and non-inoculation zone were set up for a total of 6 treatment zones. About phosphoric acid and potassium, it applied so that it might become 5 kg / 10a in a customary area and a fertilizer reduction area, and neither was given to the non-fertilization area. The pots were randomly placed in the glass room of the Tokyo University of Agriculture and Technology, Faculty of Agriculture, 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. In addition, as a chemical analysis item, the dried plant body was subjected to plant nutrient analysis and ¹⁵ N natural abundance measurement as described below.

(Field test)
The inoculation test in the field was conducted in two paddy fields.

  One place is the FM Honmachi paddy field (Tama River alluvial soil) attached to Tokyo University of Agriculture and Technology. Here, there are three fertilization stages: the conventional section (10 kg / 10a), the reduced fertilizer section (7 kg / 10a) with 30% nitrogen reduction, and the non-fertilized section (0 kg / 10a). A ward / non-inoculation zone was set up for a total of 6 treatment zones. About phosphoric acid and potassium, it applied so that it might become 10kg / 10a in a customary area and a fertilizer reduction area, and neither was given to the non-fertilization area.

Rice “leaf star” seedlings grown for 20 days were transplanted at 3 plants per plant so that the planting density was 22.2 strains / m ² .

  The survey items were 1), 2) and 3) below.

  1) As growth survey items, five continuous strains from within the community of each treatment section 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 for the measurement of 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. 8 strains (16 strains in total) was collected from each treatment section, and the above-ground fresh weight was measured. Among them, 4 strains having an average fresh weight were classified according to the site 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) As chemical analysis items, dry matter was subjected to plant nutrient analysis and ¹⁵ N natural abundance measurement described below.

  The other is a paddy field in Ogata Village, Akita Prefecture. Here, the seedling box was immersed in a solution in which the inoculum was suspended 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)
For nitrogen inoculum was fixed ascertain has shifted toward the leaves star, a further portion of the drying plant powder obtained in pot tests and field trials treated with ultrafine pulverizer, ^{15 N} natural It used for the measurement of abundance ratio. 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. In the root part, when the non-inoculated area of the customary area is set to 100 in the index display, the inoculated area becomes 205, and the dry weight of the root amount increased by about 2 times by the inoculation.

  Further, even in the reduced fertilizer group, an index of 137 was shown compared to the conventional non-inoculated group, and the root dry matter weight tended to increase, and it was found that inoculation with the 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 to be more expressed.
The value of total dry matter showed a significant increase with nitrogen fertilization and 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 (no inoculation) plot. It was. The value of total dry weight in the reduced fertilizer inoculation area was similar to that in the conventional non-inoculation area, suggesting that the reduced nitrogen content could be compensated by inoculation. Fig. 1 shows the correlation between fertilization stage and dry weight. As a result, there was a high correlation between the fertilization stage and dry matter weight, and the inoculation amount increased by inoculation.

(Nutrient analysis)
As a result of analysis of variance, a significant difference was observed in the fertilization stage and the effect of each inoculation (P = 0.05, P = 0.01) regarding the amount of nitrogen accumulation in the leaf star per pot. Showed that nitrogen absorption was promoted (Table 1).

  Regarding nitrogen and potassium accumulation, similar to nitrogen, significant differences were observed in the stage of fertilization and the effect of each inoculation (Table 1), indicating that phosphorus and potassium absorption was promoted by fertilization and inoculation. It was done.

(Origin of accumulated nitrogen based on δ ¹⁵ N value)
^{As for} the natural abundance ratio of ¹⁵ N, the ratio of ¹⁵ N is high in soil, and the atomic weight of nitrogen is heavy. On the other hand, the atomic weight of nitrogen in air has a low ratio of ¹⁵ N, and the atomic weight becomes light. Based on this principle, the chemical fertilizer nitrogen and the nitrogen fixed by the biological nitrogen fixation action of microorganisms have a lighter atomic weight than the nitrogen atoms derived from soil present in the soil. Therefore, the atomic weight of nitrogen in the plant body that mainly accumulates chemical nitrogen fertilizer and biological nitrogen fixed nitrogen is reduced. On the other hand, the atomic weight of the nitrogen of the plant body which absorbed nitrogen derived from soil becomes high.

Table 2 shows the δ ¹⁵ N values in the inoculation and non-inoculation sections of the root and foliage in the pot test. As a result, it was found that as the application of chemical fertilizer increased, the δ ¹⁵ N value decreased and became lighter in the non-inoculated group than in the non-fertilized group. 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 the rice inoculated with the TUAT1 strain absorbed a greater amount of nitrogen in the soil together with the chemical nitrogen fertilizer by rooting than in the non-inoculated area.

(Field test 1 (Agricultural and industrial paddy field))
(Dry weight)
Table 3 shows the measured values of dry matter weight by leaf portion of the leaf star during the ripening period. As a result of analysis of variance, 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 comparison at each fertilization stage, the inoculation group significantly exceeded the control (no inoculation) 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 factors of fertilization level and inoculation (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 the multiple comparison among all six treatment groups, there was no significant difference between the reduced fertilizer-inoculated group and the conventional non-inoculated group. Was suggested.

(Nutrient analysis)
Table 3 shows the values of nitrogen, phosphorus and potassium accumulation in leaf stars during the ripening period. For each nutrient, significant effects of both fertilization and inoculation factors were observed for the entire 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.

  At all fertilization stages, inoculation was shown to increase the nitrogen accumulation in the aerial part, and the increase was 17% in the non-fertilized area, 34% in the fertilized area, and 37% in the conventional area. The pot test showed no effect of bacterial inoculation on the nitrogen concentration in the plant, but the field test showed a clear effect of the bacterial 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 pot test results, the promotion of nitrogen accumulation in the plant body by inoculation in the field test occurs by increasing the nitrogen concentration in the plant body in a form that further increases the amount of fertilization. It was.

In addition, as with nitrogen, there is a significant effect of fertilization and inoculation on phosphorus and potassium accumulation, and for the whole plant, significant increase in accumulation by inoculation in the reduced fertilizer and customary areas for phosphorus and in the conventional area for potassium. 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))
In Ogata Village, Akita Prefecture, “Environmentally Creative Farming” is practiced, and JAS organic cultivation and pesticide-free chemical fertilizer cultivation are actively conducted. Rice cultivation is a large-scale mechanized cultivation. Therefore, it was verified whether inoculation with Bacillus genus biofertilizer is applicable to such large-scale mechanized cultivation.

  Table 4 shows the growth situation in the farmer's field.

  Regarding the plant height, there was no significant difference between the control (no inoculation) group and the inoculation group for the rice in the F and T fields.

Regarding the SPAD value of the 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 control (non-inoculated) section, and after the earliest sunrise on August 14 Even so, the number of stems was higher in the inoculated group than in the control (non-inoculated) group. Although no data is shown below, other field tests have shown similar effects.

  Table 5 shows the effect of the lower inoculation of TUAT1 on the farm field in Ogata Village, Akita Prefecture, on the yield and yield components of the rice cultivar “Komachi”.

  In the F field, there was no significant difference in the weight of the seeds and the ripening rate between the control group (TUAT1 non-inoculation) and the inoculation section, and the number of ears was rather decreased. However, the number of spikes in the inoculated group increased by 22.4% from that in the control group, and as a result, the yield of the inoculated group increased by 32.8%. In addition, in the T field, there were no significant differences in the thousand grain weight, ripening rate, and number of spikelets between the control group and the inoculation section, but the number of ears in the inoculation group increased by 14.6% compared to that in the control group. As a result, the yield of the inoculated group increased by 17.9% compared with that of the control group.

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

  The inventors of the present application are effective in expanding the root area and promoting nutrient absorption by the Bacillus pumilus TUAT1 strain (NITE BP-1356) described above, particularly in enhancing the root area and applying nutrient absorption when applied to rice. The present invention has been completed by further research on Bacillus pumilus, which pays attention and shows the same tendency.

  The inventors of the present application subsequently examined the effect of differences in the growth stage of TUAT1 strain (NITE BP-1356) used as an inoculation source on the growth of rice and the root colonization of the inoculated strain.

  In order to investigate the number of colonized bacteria in the root of rice, TUAT1 strain streptomycin 100 ppm and rifampicin 100 ppm double drug resistant mutant strain (TUAT1SR8 strain) were used for the inoculation test.

  As the variety, Hinohikari was used.

  In order to compare the growth stage of the bacteria used as the inoculation source, the test group provided a vegetative cell inoculation group and a spore inoculation group.

  The vegetative cells were collected using a Trypticase Soy broth medium at 30 ° C. under dark conditions at 105 rpm for 24 hours, and then resuspended in sterile water as an inoculum.

  Spores were collected using Trypticase Soy broth medium at 30 ° C. under dark conditions, 105 rpm, and shake culture for 10 days, suspended in physiological saline, and then killed by rinsing twice at 65 ° C. for 30 minutes. Then, the spore was resuspended in sterilized water to make an inoculum.

  Seeds were seeded with salt water, sterilized with hot water at 60 ° C. for 10 minutes, soaked at 15 ° C. for 7 days, sown at 25 ° C. for 16 hours, and sown for 20 days.

  For inoculation, the soil attached to the roots of the seedlings was removed, and the roots were immersed in an inoculation source of 1 ml per seed for 24 hours at 25 ° C. under dark conditions.

  The control (no inoculation) group was immersed in sterilized water in the same manner. Inoculated seedlings are transplanted as three plants per plant in a semi-transparent pot filled with 400 ml of non-fertilizer medium supplemented with 0.5 g of Rin Kaan 14 and managed in a glass greenhouse at a minimum night temperature of 15 ° C for 33 days. did.

  The survey items were the rice growth and the TUAT1SR8 strain fixability in the rice root.

  In the fixability investigation, the soil adhering to the root was washed with tap water, and then a solution shaken in physiological saline for 10 minutes was used as a root surface sample. Further, a root was taken out from the physiological saline, and a solution obtained by chopping, grinding, and diluting with physiological saline was used as a sample in the root.

  The number of bacteria was measured by a dilution plate method using Trypticase Soy agar containing 100 ppm rifampicin, 100 ppm streptomycin, and 200 ppm cycloheximide.

  As a result of the growth survey, the root weight was significantly increased by 42% in the vegetative cell inoculation group and 80% in the spore inoculation group.

As a result of the investigation of colonization, the number of colonized bacteria at the time of transplantation immediately after the treatment for 24 hours in the vegetative cell inoculation group was 10 ⁴ (cfu / groot), but it was not detected after 7 days. It was revealed that the number of colonized bacteria at the time of transplantation was 10 ⁷ (cfu / groot) and 10 ^{4 to 5} (cfu / groot) even after 25 days from transplantation, and the colony was stably settled in the rice root.

  In order to investigate the spore ratio of the fungus that settles at the root, after preparing the root surface and the sample in the root, roasting at 65 ° C. for 1 hour to kill vegetative cells, it was reduced to about half It was considered that trophoblast cells and spores were mixed at the same rate.

  This suggests that the spores are not in a dormant state but exist as one stage of the growth ring of this strain at the root.

  This study suggests that by using spores as the inoculum, it is possible that the rooting of rice will be more stable and the root weight increase effect may be stabilized. The possibility that it is useful is confirmed.

  The present invention based on these studies is a biofertilizer (microbe fertilizer) having a spore ratio of 100%, which is obtained by culturing Bacillus bacteria and then heat-treating at 65 ° C to 80 ° C for 60 minutes or more.

  Since the microbial fertilizer of the present invention is obtained by culturing Bacillus genus bacteria to a spore ratio of 100%, it can be stored for a long period of time at room temperature (around 25 ° C.). 6 months or more). In addition, when applied, the roots of crops (especially rice) are improved.

  According to the study by the inventors, it has been confirmed that the increase in the amount of growth of the crop (particularly rice) is higher than the non-inoculation or vegetative type inoculation by spore formation.

  It has been conventionally known that stress tolerance such as temperature and moisture can be enhanced by forming spores. The inventors of the present application, by setting the spore rate to 100% under the above-described conditions, can more effectively express characteristics such as growth promotion conventionally recognized in Bacillus bacteria, and in particular, applied crops ( For example, the present invention has been completed by finding out that the root area expansion of rice), the fixability to roots, the effect of increasing the weight of roots, and the effect of promoting nutrient absorption are improved.

  As described above, after culturing the Bacillus genus bacteria, the trophoblast can be completely killed by heat treatment for 60 minutes or more in a temperature range of 65 ° C. to 80 ° C., and the spore ratio can be 100%.

  According to the study by the inventors, the vegetative type is completely killed to a spore ratio of 100%, and then the root area of the applied crop is expanded, the root establishment is improved, the root weight is increased, the nutrient is absorbed. In order to effectively improve the accelerating effect and the like, it was desired to perform heat treatment for 60 minutes or more in a temperature range of 65 ° C to 80 ° C.

  Note that the heat treatment time of 60 minutes or more may be divided into a plurality of times, and the total heat treatment time may be 60 minutes or more.

  In addition, with a spore ratio of 100%, it is possible to effectively improve the root area of applied crops, improve root fixation, increase root weight, promote nutrient absorption, etc. In view of the above, it is desirable that the heat treatment in the temperature range of 65 ° C. to 80 ° C. does not exceed 70 minutes in total.

  The conditions for spore formation with a spore ratio of 100% are not particularly limited as long as the conditions for the heat treatment for 60 minutes or more in the temperature range of 65 ° C. to 80 ° C. are satisfied. For example, the cells are cultured on a Trypticase soy plate (20 ml medium, 9 cm diameter petri dish) at 28 ° C. for 10 to 14 days. Bacteria are collected from here, washed with physiological saline (3 times), collected again and suspended in physiological saline, and then heat-treated at 65 ° C. for 30 minutes, by repeating the above steps twice. The heating time at 65 ° C. is 60 minutes. The biofertilizer (spore fluid) of the present invention thus obtained is refrigerated at 4 ° C., and the stored biofertilizer (spore fluid) is diluted with sterilized water and used as an inoculation source.

The above-described biofertilizer of the present invention has a fungus concentration of 1. from the viewpoint of effectively improving the root area of applied crops, improving root fixation, increasing root weight, promoting nutrient absorption, and the like. 0 × is desirably ^{10 7 ~9.0 × 10 7 cfu /} g.

  In the biofertilizer of the present invention described above, the Bacillus genus bacterium can be Bacillus pumillus TUAT1 strain. The Bacillus pumillus TUAT1 strain is a bacterium that has been deposited internationally at the Patent Microorganism Depositary under the accession number NITE BP-1356. According to the study by the inventors, the spore rate is set to 100%. It was possible to effectively improve root area expansion, root anchorage improvement, root weight increase effect, nutrient absorption promotion effect, and the like.

  Next, the granular biofertilizer (granular microbial fertilizer) proposed by the present invention is formed by immersing granular fertilizer materials in the above-described biofertilizer of the present invention, and then drying it until the water content becomes 12% to 2%. Is. Or after adding and mixing the bio-fertilizer of this invention mentioned above to the fertilizer material and granulating, it dry-processes until it becomes 12%-2% of water | moisture content.

  The drying process here is performed for the purpose of moisture adjustment.

When the bacterial concentration in the granular biofertilizer of the present invention is 1.0 × 10 ^{7 to} 9.0 × 10 ⁷ cfu / g as described above, it is desirable that the drying treatment is performed until the water content becomes 2%, When the drying process is finished in a state where the water content exceeds 15%, even if the bacterial concentration of the granular biofertilizer at the time of manufacture is in the range of 1.0 × 10 ^{7 to} 9.0 × 10 ⁷ cfu / g In the subsequent storage, the number of bacteria is less than 10 ⁷ cfu / g, which is not preferable.

In addition, in order to set the bacterial concentration of the granular biofertilizer after the drying treatment to 1.0 × 10 ^{7 to} 9.0 × 10 ⁷ cfu / g, in the drying process performed for the purpose of moisture adjustment, However, it is desirable to perform ventilation drying or fluidized bed drying. For example, the moisture can be adjusted to 12% to 2% by drying within 4 hours using a ventilation dryer.

  By using the above-described microbial fertilizer of the present invention as a granular biofertilizer in this way, the number of bacteria and the effect thereof can be maintained for a longer period of time (long-term storage at room temperature for one year or more is possible). Moreover, handling property improves and it applies as a granular fertilizer (for example, it mixes and applies to culture soil at the time of seedling raising), the fixability to the root of a crop can be improved further, and a fertilization effect can be improved.

  The fertilizer material may be any one or a combination of diatomaceous earth, zeolite, and silica gel.

  Silicic acid material has improved lodging resistance and healthy seedling effect of rice. Therefore, by using silica gel as a fertilizer material, or by using silica gel as a part of fertilizer material, increase the production / reduction in rice production. The effect of reducing fertilizer can be improved.

  These fertilizer materials are granulated into granular fertilizer materials, which are dipped in the biofertilizer of the present invention described above, and then dried and sieved to obtain granular biofertilizers having an average particle size of 2 to 4 mm. .

  Or after adding and mixing the biofertilizer of this invention mentioned above to these fertilizer materials and granulating, it is dried and sieved and it is set as the granular biofertilizer with an average particle diameter of 2-4 mm.

  The granulation can be performed in various granulation formats regardless of the granulation format such as rolling granulation (drum, dish), extrusion molding (pellet), compression molding (briquette) and the like.

  Even if it is the granular biofertilizer manufactured in any form mentioned above, it is desirable from the viewpoint of handling property that the hardness is 1 kgf or more.

  The fertilizer material in the granular biofertilizer of the present invention described above preferably includes a porous material having a pore diameter of μm or more. In the biofertilizer of the present invention, the size of the microorganism is 1 μm or more. Therefore, when a porous material having a pore diameter of μm or more is used, it is advantageous because microorganisms can be enclosed in the pores of μm or more.

  For example, diatomaceous earth (unfired) is a porous material having a pore size of μm or more, and is therefore useful as a porous material for encapsulating Bacillus spp.

  In the case of using a combination of the above-mentioned various types of fertilizer materials, for example, silica gel and diatomaceous earth (not fired) are used in a ratio of 7: 3 by weight, and the total weight is 1.2. Add ~ 3% binder (eg starch), mix and mold (granulate). On the other hand, after culture | cultivating Bacillus genus bacteria, the solution of the biofertilizer of this invention which was heat-processed in the temperature range of 65 to 80 degreeC for 60 minutes or more and made the spore rate 100% is prepared. And the granular fertilizer material prepared as described above is added in an amount equal to the biofertilizer solution and immersed for 1 hour. Thereafter, the biofertilizer solution can be removed by decantation and dried by ventilation (70 ° C., 4 hours) to obtain the granular biofertilizer of the present invention having a moisture content of 2 to 12%.

  In addition, in the above, a binder (for example, for example, added in a weight ratio of 1.2 to 3% with respect to the total weight of the fertilizer material composed of any one or a combination of diatomaceous earth, zeolite, and silica gel) Starch) is added for the purpose of allowing the applied granular biofertilizer of the present invention not to disintegrate immediately but to disintegrate in about one week after application. If it is a binder for fertilizers used for such a purpose, various things well-known in this technical field can be used.

  Hereinafter, preferred embodiments of the present invention will be described. However, the present invention is not limited to the above-described embodiment and examples described below, but within a technical scope grasped from the description of the scope of claims. Various changes can be made.

  Bacillus pumillus TUAT1 strain (NITE BP-1356), which is a genus of Bacillus, can take a form called a spore in its life cycle and can have a strong resistance to environmental conditions such as temperature and moisture. is there. Moreover, the TUAT1 strain has a function of promoting root elongation in rice, and can contribute to an increase in the acquisition of nutrient absorption, thereby increasing the yield.

  However, to date, it has not been clear how much the difference between vegetative type and spore type affects these effects. Therefore, in this example, it was clarified whether inoculation of rice with different forms of TUAT1 strain affects the growth of rice.

  In this example, in addition to the TUAT1 strain, an antibiotic-resistant strain (TUAT1SR8 strain) in which rifampsin and streptomycin resistance were imparted to TUAT1 was used for the purpose of appropriately evaluating rice root fixation.

Adjustment of inoculum The adjustment of vegetative type and spore inoculum was performed as follows.

The vegetative inoculation source was prepared by adjusting two TUAT1 strains stored in 50% glycerol solution [TUAT1 strain and the above-mentioned anti-substance resistant strains (TUAT1SR8 strain) imparted with resistance to streptomycin and 500 ml of TUAT1 strain]]. 200 ml Trypticase Soy Broth (manufactured by BD) was inoculated using a flask. After inoculation, the cells were cultured for 24 hours at 28 ° C. and 110 rpm. After culturing for 24 hours, the cells were collected by a centrifuge (Centrifuge Avanti TM25 manufactured by BECMAN) and washed three times using the same amount of physiological saline. After washing, the inoculation source was adjusted to a concentration of 10 ⁷ cfu / ml with sterilized water. The inoculum was stored refrigerated at 4 ° C. until use.

The inoculation source of the spore was inoculated into 20 ml Trypticase Soy Agar prepared by using two types of TUAT1 strains (TUAT1 strain and TUAT1SR8 strain described above) using a 9 cm diameter petri dish. After inoculation, the cells were cultured at 28 ° C. After culturing, the cells were collected by a centrifuge and washed three times using the same amount of physiological saline. After washing, heat treatment was performed at 65 ° C. for 30 minutes to kill vegetative cells. Thereafter, washing and heat treatment were performed once again, and the inoculum was adjusted to a concentration of 10 ⁷ cfu / ml with sterilized water. The inoculum was stored refrigerated at 4 ° C. until use.

Cultivation test Test plant Rice (Oryza sative) cv. Hinohikari seeds overview Hinohikari seeds were soaked for 8 days at 15 ° C. after salt water selection and hot water disinfection. After soaking, the half-day sprouting-treated rice was sown in seedling culture soil (non-fertilizer seedling culture soil: fertilizer-containing seedling culture soil = 2: 1). After washing roots of seedlings grown for 20 days with tap water, 1 ml of TUAT1 strain and TUAT1SR8 strain each adjusted to a concentration of 10 ⁷ cfu / ml were inoculated in 1 ml per different test area. Twenty-four hours after inoculation, the seedlings were transplanted into a 500 ml pot filled with 400 ml of non-fertilizer breeding seedling culture soil and 0.5 g of Linkaan No. 14 added thereto. Three transplants were made per pot. After transplanting, the plants were cultivated in a glass house, and after 33 days, the above-ground and root weights of rice inoculated with TUAT1 strain and TUAT1SR8 strain were measured.

Monitoring of the number of colonized bacteria Only the rice seeded with the TUAT1SR8 strain was used to monitor the number of colonized bacteria in the root surface and root.

1) Root surface sample Rice grown in a glass house was sampled after 0, 4, 7, and 14 days. The soil attached to the sampled roots was removed, and a solution obtained by shaking the sample with physiological saline about 50 times the weight of the root for 30 minutes was used as a root surface sample.
2) Root sample After preparing the root surface sample, an appropriate amount of the root was ground with physiological saline. After grinding, a suspension obtained by adding about 10 times the weight of the physiological saline was used as a root sample. 3) Counting the number of bacteria Using a trypticase soy plate containing rifampicin 100 ppm, streptomycin 100 ppm, cycloheximide 200 ppm The number of bacteria was measured by the dilution plate method.

Investigation of the spore rate The prepared inoculum, root surface and root sample were heat-treated at 65 ° C. for 1 hour. The ratio was calculated with the number of viable bacteria before treatment and the number of spores after treatment.

Results FIG. 2 is a reference photograph showing the results of the study in this example, in which the left side is a spore type and the right side is a vegetative type.

As a result of examining the influence of the vegetative type of Bacillus pumilus TUAT1 strain and the inoculation source of spore on colonization in rice roots, the number of vegetative type was 4.6 × 10 ⁴ cfu / What was detected in ml was shown not to be detected after 7 days. Similar results were obtained at the root surface.

On the other hand, the number of spores in the roots was detected at 6.3 × 10 ⁷ cfu / g immediately after inoculation, and even after 25 days, the number decreased slightly to 5.4 × 10 ⁵ cfu / g. However, the number of bacteria was shown to be maintained (Table 6).

  As a result of examining the influence on the growth of rice by the difference in vegetative type and spore inoculation source of the two types of TUAT1 strain inoculation, the root weight is 0.1% in the spore type in comparison with the vegetative type inoculation A significant increase was observed (t test).

  Moreover, in TUAT1 SR8 strain | stump | stock, it was recognized that the root weight and the number of tillers increased significantly (Table 6). From these results, it became clear that the difference in the inoculation source of the TUAT1 strain is that the spore has a higher effect on rice colonization and growth.

From the viewpoint of the effect on the growth of rice, such as the root colonization improvement and the root weight increase effect, the TUAT1 strain and the antibiotic-resistant strain (TUAT1SR8 strain) are compared with the vegetative type. It was confirmed that the same excellent effect was exhibited. Thus, as to the action / effect and mechanism expressed by the biofertilizer of the present invention, the same results were obtained as confirmed in this example, regardless of whether the TUAT1 strain or the TUAT1SR8 strain was used. It is acknowledged to be shown.

Antibiotic resistant strain (TUAT1SR8 strain), which gave rifampsin and streptomycin resistance to TUAT1 strain, was inoculated into 200 ml of Trypticase soy broth liquid medium and cultured at 30 ° C. and 110 rpm for 11 days. After culturing, heat treatment was performed twice at 65 ° C. for 30 minutes to prepare 5.4-5.5 × 10 ⁸ cfu / ml spores.

  The heat treatment was performed by bathing, and after the treatment, the sample was ice-cooled. After the heat treatment, the number of bacteria was counted by a dilution plate method. The medium for counting the number of bacteria was Trypticase soy plate containing 100 ppm of streptomycin and rifampsin. The count of the number of bacteria was calculated as an average value of 2 samples in duplicate.

Results The results of the study are shown in Table 7 below.

In the spore of TUAT1SR8 strain, a decrease in the initial number of bacteria was hardly observed even at 180 ° C. under the heat treatment at 70 ° C. It was shown to decrease to a bacterial count concentration of 1000. From this, it became clear that the heat resistance of the TUATSR8 strain was at 70 ° C.

  The effect of the material which enclosed TUAT1 stock in granular fertilizer was examined.

Granular fertilizer Silica gel and diatomaceous earth (unfired diatomaceous earth) are mixed at a weight ratio of 7: 3, and granulated fertilizer (added starch with a total weight of 3% as a granulation accelerator (binder)).

Powdered fertilizer Preparation of fertilizer TUAT1 stock encapsulated material mixed with silica gel and diatomaceous earth in a ratio of 7: 3 (addition of starch with a total weight of 3% as a granulation accelerator (binder)) Preparation of bacterial solution Rifampsin in TUAT1 strain And 10 antibiotic-resistant strains (TUAT1SR8 strain) to which streptomycin resistance was imparted were inoculated into 10 Erlenmeyer flasks containing 200 ml Trypticase soy broth liquid medium, and cultured at 28 ° C. and 110 rpm for 11 days. After the culture, heat treatment was performed at 65 ° C. for 1 hour, and centrifugation was performed at room temperature at 8,000 rpm for 10 minutes. After centrifugation, it was washed 3 times with physiological saline, suspended in 2 liters of physiological saline, and adjusted to a bacterial concentration of 8.7 × 10 ⁸ cfu / ml.

Preparation of formulation 200 g of granular fertilizer and powdered fertilizer were placed in a stainless steel vat sterilized with 70% ethanol, 200 ml of the prepared bacterial solution was added, and immersion treatment was performed for 1 hour. After the treatment, drying was performed using a ventilator at 70 ° C. for 4 hours. After drying, it was refrigerated at 4 ° C. until used for each test.

Summary of inoculation test Hinohikari seeds were soaked for 8 days at 15 ° C. after salt water selection and hot water disinfection. After soaking, the half-day germinated fir was sown in a seedling culture soil (Lovery: Spring-style culture soil: 2: 1). Germination was performed at 28 ° C. in the dark for 2 days, and seedlings were grown in Groth Cabinet at 23 ° C. As for seedling conditions, a quarter-sized seedling vat was used, and after filling 900 ml of soil per vat, 300 g of water was irrigated, 25 g of germinated fir were covered with 200 ml of soil (non-fertilizer). The seedlings were transplanted into a 500 ml disposable cup filled with 400 ml of non-fertilizer raising seedling culture soil and 0.5 g of Rinkaan No. 14 added. Three transplants were made per pot. After transplanting, the plants were cultivated in a test greenhouse, and the dry weight of the above-ground part and the root part at the time of transplanting and the plant height, tillering, dry weight of the above-ground part and the root part after two months were measured. The values at the time of transplantation were the average per 100, and 2 months later, 1 ward was 6 pots, 4 stations, and the values were the average per pot.

The results are shown in Table 8.

  In Example 3, a bacterial solution immersion method (dry treatment until the moisture content becomes 12% to 2% after the granular fertilizer material was immersed in the microbial fertilizer of the present invention) was adopted in the preparation. In this example, in order to examine an efficient production method, different production methods (after adding and mixing the microbial fertilizer of the present invention to the fertilizer material and granulating, drying treatment until the water content becomes 12% to 2%) The number of bacteria in the preparation prepared in (1) was examined.

In the same manner as in Example 3, a bacterial solution having a bacterial concentration of 8.7 × 10 ⁸ cfu / ml was prepared.

Fertilizer materials similar to Example 3 (silica gel and diatomaceous earth (unfired diatomaceous earth) were mixed at a weight ratio of 7: 3, and granulated molded fertilizer (total weight 3 as a granulation accelerator (binder)) % Starch was added), and an equal amount of the bacterial solution (8.7 × 10 ⁸ cfu / ml) was added and mixed to granulate.

  After granulation, the mixture was dried at 70 ° C. for 2 hours and 30 minutes using a ventilation dryer.

  The obtained preparation was ground in a mortar, 45 ml of sterilized water was added to about 5 g and shaken for 10 minutes, and the number of bacteria was counted by a dilution plate method.

  The results are shown in Table 9.

In the preparation obtained in this example, it was shown that the same number of bacteria as in Example 3 can be obtained.

  The preferred embodiments and examples of the present invention have been described above, but the present invention is not limited to the above-described embodiments and examples, and variously within the technical scope grasped from the description of the claims. It can be changed.

Claims (6)

A method of producing a biofertilizer having a spore rate of 100% obtained by culturing Bacillus pumillus TUAT1 strain (NITE BP-1356) , which is a bacterium belonging to the genus Bacillus, and then heat-treating at a temperature range of 65 ° C to 80 ° C for 60 minutes or more. The method for producing a biofertilizer according to claim 1, wherein the fungus concentration is 1.0 x 10 ^{7 to} 9.0 x 10 ⁷ cfu / g. After immersing the fertilizer material of granular biofertilizer produced by the production method of biofertilizer according to claim 1 or 2, wherein a method of producing a granular biofertilizer obtained by drying until 12% to 2% moisture. After granulation was added and mixed biofertilizer produced by the production method of biofertilizer according to claim 1 or 2, wherein for materials fertilizer, granular biofertilizer obtained by drying until 12% to 2% moisture How to manufacture . The method for producing a granular biofertilizer according to claim 3 or 4, wherein the fertilizer material is composed of any one or a combination of diatomaceous earth, zeolite, and silica gel . The method for producing a granular biofertilizer according to any one of claims 3 to 5, wherein the fertilizer material includes a porous material having a pore diameter of not less than µm .