JPS5950311B2 - Active rhizobial bacteria formulation - Google Patents

Description

[Detailed description of the invention] The present invention relates to active rhizobia.

Legumes form root nodules in symbiosis with rhizobia, fix nitrogen in the air, and can increase productivity even in barren land.

We have already achieved results in increasing the production of leguminous plants by mass culturing rhizobia and artificially inoculating the cultured rhizobia.

That is, after culturing rhizobia, the bacteria are collected by centrifugation.

This is adsorbed onto adsorbents such as soil, vermiculite, Kanuma soil, their fine porous stones, and plastic materials, and the moisture content is 40 to 5.
It was stored in a bag at around 0%, and when necessary, it was mixed with leguminous plant seeds and inoculated.

However, these rhizobia become deactivated in just three months, and if they are not deactivated, their activity is reduced, so when applied to cultivated land, they are suppressed by other microorganisms, and the leguminous plants are unable to establish root nodules, resulting in reduced productivity. Improvements are often not achieved.

Therefore, cultured rhizobia must be applied within three months after collection, which is unsatisfactory for large-scale practical application.

Therefore, the present inventor has been engaged in research for a long time in an attempt to solve the above-mentioned problem of inactivation or decreased activity of rhizobia during storage by activating rhizobia. succeeded in becoming

In other words, we have obtained new knowledge that rhizobia can be activated by mixing rhizobia and photosynthetic bacteria.

The present invention is based on this new knowledge, has the effects as shown in the test examples (described later), and has enabled large-scale practical application.

That is, the present invention is a composition of active rhizobia, which is a mixture of rhizobia and photosynthetic bacteria.

After one year, the active rhizobia formulation according to the present invention
Not only does it remain active even when applied to cultivated land, but
Even in the soil, due to the protective effect of photosynthetic bacterial body secretions, nodules are easily established on leguminous plants without being dominated by other microorganisms in the soil, and the nitrogen supply to leguminous plants is not sufficient. , it is possible to grow healthy and improve its productivity.

The present invention will be explained in detail below.

Rhizobia are cultured, for example, in the following culture medium.

(1) Mannit 10? , K2HPO40,5fi
, Mg5O2-7H200,2P, NaCtO21
1. CaCO23V, yeast extract 1v, distilled water 1t...
...pH 6,8 (2) Mannitol 10f%KNO2
0,5fi, K2 HPO40,5ri, Mg S 0
4 ・7 H200, 2V, Na Cto, 2? ,
Traces of MnSO4-5H20, traces of FeCl2, distilled water 1
t%...)...pH 6,8 Photosynthetic bacteria are cultured, for example, in the following culture medium.

(NH4) 25040.3 fI, K per 1 ton of water
H2PO40,5f, Mg504-7H200,2S'
, NaCtO, 5P, Ca Cl2O, 5V, NaHC
A basal medium was prepared by mixing 0.01 g of O30.2P and yeast extract, and 0.4 g of Na2S-9H20 was added to this.
% and adjusted the pH to 8.4 with KOH solution.

In addition, when culturing a mixture of rhizobia and photosynthetic bacteria, the above-described culture medium for rhizobia is used.

Examples of rhizobia include soybean bacteria: Rhizopium japonicum (R
hizobium japonicum) IFONo1
3338, Adzuki bean fungus: Rhizopium melilotsutei (Rh
i zobi ummeliloti) IFONo,
, 13336, Clover fungus: Rhizobium trifolii I
FONo, 13337 and other common root nodule bacteria can be used.

Examples of photosynthetic bacteria include Rhodopseudomonas capsulatus.
Rhodospirillum rubrum (Rhodospirillum rubrum)
) Microtechnical Research Institute No. 878, Chromatium pinosum (C
hromatium vinosum) Microtechnical Laboratory Bacterium No. 8
No. 90, Rhodopseudomonas suphaeroides (Rh
odopseudomonas 5pheroides
) IFO No. 12203, other photosynthetic bacteria can be used.

The mixing ratio of rhizobia and photosynthetic bacteria is usually 1:1, and there is no strict mixing ratio; as long as the two are mixed, rhizobia will be activated.

Rhizobia and photosynthetic bacteria may be mixed by culturing them individually and collecting them, or by culturing them together.

In the case of monoculture, the collected bacterial cells are mixed with rhizobia and photosynthetic bacteria and then adsorbed to soil or other adsorbent, or they are air-dried or ventilated without adsorption to retain moisture. Dry the material to 20-30%.

In a mixed bacterial cell of rhizobia and photosynthetic bacteria, the rhizobia are activated, and even if stored at room temperature for more than a year, the nodule microbial ability hardly decreases, and root nodules are reliably formed, increasing the productivity of leguminous plants. Improve.

The reason why the mixture of rhizobia and photosynthetic bacteria improves granulation activity and shelf life, and also why it does not lose its activity when dried, is currently under investigation.

Examples of the present invention are shown below.

Example 1 The above (1) mannitol medium was placed in an Erlenmeyer flask, and Rhizopium japonicum and Rhodopseudomonas capsulatus were inoculated at a ratio of approximately 1:1 in number of bacteria, and aerated culture was carried out for 2 days at 30° C. under 3000 lux lighting. Then, collect the cultured bacteria using a centrifuge.

The collected bacterial cells were dried with ventilation at 25''-30°C until the moisture content was 30% to obtain a mixture of active rhizobia.

Example 2 A conical flask was filled with mannite medium (above), and inoculated with Lithopum melilotsutei and Rhodopseudomonas phuphaeroides at a ratio of approximately 2:1, and incubated at 30°C.
Aerated culture was performed for 2 days under 3000 lux lighting, and the cultured bacteria were collected using a centrifuge.

The collected bacterial cells were dried with ventilation at 25° C. to 30° C. until the moisture content was 80% to obtain a mixture of active rhizobia.

Example 3 Put the mannitol medium (1) above into an Erlenmeyer flask, inoculate Rhizopium trifolii and Rhodospirillum rubrum at a ratio of approximately 1:1 in terms of bacterial count, and heat at 30°C and under 300° lighting.
Aerated culture was carried out for 2 days at 0 lux, and the cultured bacterial cells were collected using a centrifuge to obtain a mixture of active rhizobia.

Example 4 The above (2) mannitol medium was placed in an Erlenmeyer flask, Rhizopium japonicum was inoculated, cultured under aeration at 30°C for 2 days, and the cultured cells were collected using a centrifuge.

On the other hand, the culture solution of the photosynthetic bacteria described above was stored in a closed, illuminated culture tank (the inside of the tank was evenly illuminated with fluorescent lights placed at equal intervals around the outer circumference of a transparent glass cylinder, and a stirrer was installed inside the tank). The culture solution was inoculated with Rhodopseudomonas capsulatus and incubated at 30°C.
The cells were cultured for 1 day under illumination of 1000 lux with stirring at a rotational speed of 13 times per minute, and the culture solution was collected using a centrifuge.

Next, the Zopium japonicum obtained above and Rhodopseudomonas capsulatus were thoroughly mixed in a volume ratio of approximately 1:1, and then air-dried at 25°C to 30°C for one week to a moisture content of 80%. A blend of active rhizobia was obtained.

Example 5 In an Erlenmeyer flask, add 0.2 Vt of malic acid (potassium salt) to the above-mentioned (2) mannitol medium, inoculate Rhizopium melilotsutei and Chromatium pinosum at a ratio of approximately 1:1 in terms of number of bacteria, and heat at 30°C. , 3,000 lux for 2 days, and the cultured cells were collected using a centrifuge.

The collected bacterial cells were air-dried at 25° C. to 30° C. until the moisture content was 80% to obtain a blend of active rhizobia.

Test examples for demonstrating the effects of the present invention are shown below.

Test Example 1 Put the mannitol medium (1) above into an Erlenmeyer flask, inoculate with Lisopium japonicum, culture at 30°C for 3 days, and collect the cultured bacteria using a centrifuge.

The collected bacterial cells were dried with ventilation at 25°C to 30°C until the moisture content was 30%.

When the number of viable Zopium japonium bacteria was counted one week after drying, the number of viable bacteria was about 10% of the number one week before.

On the other hand, the active rhizobia mixture obtained in Example 1 was dried and
When the number of viable Zopium japonicum bacteria was counted after a week, the number of viable bacteria was about 85% of the number one week before.

Test Example 2 Place the mannitol medium (1) in an Erlenmeyer flask, inoculate Rhizobium melilotsutei, culture at 30°C for 2 days, and collect the cultured cells by centrifugation.

The collected bacterial cells were dried with ventilation at 25°C to 30°C until the moisture content was 30%.

After storing this Rhizopium melilotsutei in a refrigerator at 4° C. for one year, the number of viable Rhizopium melilotsutei bacteria was counted, and the number of viable bacteria was about 0.001% of that one year ago.

On the other hand, after storing the active rhizobia mixture obtained in Example 2 in a refrigerator at 4°C for one year, the number of viable Rhizopium melilotsutei bacteria was counted, and the number of viable bacteria was about 75% of that one year ago. .

Test Example 3 Fill an Erlenmeyer flask with the mannitol medium (mentioned above), inoculate it with Rhizopium trifolii, culture with aeration at 30°C for 2 days, collect the cultured bacteria with a centrifuge, and suspend the bacteria. Adjust the liquid and apply it to 100 cultured clovers (10 mt each), about 2 kn
After that, I saw root nodule formation.

The ability to settle is weak, with some grains not settling on average, 0,
There were 8 pieces/book.

On the other hand, a suspension of the active rhizobium mixture obtained in Example 3 (10 live cells/rnt) was applied to 100 cultured clovers (10 m7 each), and after about 2 months, nodule formation occurred. I saw life.

An average of 1.5 pieces/plant, at least one seed, was attached, and the growth of the stems and leaves was about 1.5 times more promoted than when Rhizopium trifolii was applied alone.

Test Example 4 Prepare three frame test plots (sandy loam soil 1 m x 14 nm, partitioned to a depth of 1 m with plastic plates), and transplant 9 soybean seedlings to each plot.

The first area was used as a control area in which Rhizopium japonicum was not inoculated at all, and the second area was where Rhizopium japonicum was cultured alone in Example 4.
The mixture was dried to 30% moisture over 10 days and stored at room temperature for 6 months, and then inoculated. The soybeans stored for several months were inoculated and the soybean yield and number of nodules attached were investigated.

As a result, the yield was 8.58r/bottle in the first section and 4.58r/bottle in the second section.
87? / book, 3rd ward 6.05g/nodule, 1st ward 1.7 nodules/nodule (these are small nodules that are thought to be naturally infected), 2nd ward 2.1 nodules/nodule, 2nd ward 2.1 nodules/nodule, The effect of a mixture of rhizobia and photosynthetic bacteria was found, with 2.5 pieces per plant in 3 plots.

Moreover, the nodule type of the 3rd section is 1. compared to that of the 2nd section.
It was found to be 7 times higher.

Test Example 5 Recently, continuous cropping disorders have been occurring frequently in plants such as azuki beans, peas, broad beans, etc., and it is currently difficult to prevent such disorders. I tried it.

Three frame test plots (separated by continuous cropping impaired soil cloth m x 1 m and a depth of 1 m with plastic plates) are prepared, and 9 adzuki bean seeds are sown in each plot.

The first section was a control section in which Rhizopum melilotsutei was not inoculated at all, and the second section was a control section in which Rhizopum melilotsutei was inoculated into the same medium used in Example 5.
After aeration culture for 1 day, the cultured bacteria were collected using a centrifuge.
The collected bacterial cells were dried under ventilation at 25°C to 30°C until the moisture content was 30%, and dried Zopum melilotsutei stored at room temperature for one month was inoculated, and the third area was inoculated with the same method as in Example 5. Obtained, Zopium melilotsutei and Chromatium.
A mixed dry product of Pinosum that had been stored at room temperature for one month was inoculated and the yields were compared.

As a result, the first plot died and died due to the occurrence of continuous cropping-inhibiting bacteria, and the yield was 0, and the second plot did not grow well, but none died, and the yield (fresh 1 averaged 12.35 '/Book,
In the third section, there was no tendency for continuous cropping failure at all, and the plants grew smoothly, with an average yield of 33.8 g/plant.

Furthermore, in the third ward, the number of harmful Fusarium bacteria was extremely suppressed, being less than 1/10,000 of the Fusarium bacteria in the 1st ward, and less than 1/100 of the Fusarium fungi in the 2nd ward.

Claims (1)

[Claims] 1. A mixture of active rhizobia, which is a mixture of rhizobia and photosynthetic bacteria, in order to prevent the inactivation and death of rhizobia and to activate rhizobia.