KR930009509B1 - Composition of rhizobium sp for soybean nodules - Google Patents

Abstract

A Rhizobium meliloti YCKA 500 (KFCC-10725), Rhizobium meliloti YCKA 622 (KFCC-10726), Rhizobium sp. YCK 15 (KFCC-10717) and Rhizobium japonicum YCK 213 (KFCC-10728) were isolated from soil. An inoculant for higher yields of leguminous crops (peanut, soy-bean, alfalfa etc.) contains the culture broth of 4 species of the screened microorganisms with 1 or 2 species of carrier. Peanut oil, soy-bean oil or mineral oil was used as a sticker in stead of a arabian gum or xanthan gum is promoting adhesion of the inoculant to the seeds.

Description

Mycobacterium inoculum to increase the two crops

1 is a graph showing the survival rate of mycorrhizal fungi when oil is used as a seed adhesive of the two crops of mycorrhizal fungi.

Figure 2 is a graph showing the contribution of soybean growth of myococcal bacteria inoculated by oil as the mycobacterial adhesive substance.

The present invention can effectively reduce the use of nitrogenous chemical fertilizers for many years, as well as the use of excellent mycorrhizal fungi when cultivating soybean crops in farmhouses, and can significantly improve crop production, and inoculate the mycorrhizal fungi for the increase of the soybean crop. It is about.

Root fungus is an aerobic bacillus of gram-negative bacteria found in legume habitats. The scientific name is Rhizobium. Root bacteria, unlike other bacteria, invade the roots of legumes and make root nodules. As the root nodules grow, they biologically fix about 80% of the nitrogen gas in the air, providing a sufficient amount of nitrogen for the entire growth period of the plant. do. This characteristic of mycorrhizal bacteria adds a practical value, especially when cultivating legumes, which can prevent the risk of nitrate poisoning in livestock due to overuse of conventional fertilizers.

These fungi have been introduced by Lachmen (J. 1858. Ueber Konllchen der Leguminsen. Landw. Mitt, Zeitschn. K. Lehranst. Vers. Sta. Poplesdorf (Bonn) 37.) in the mid-19th century, 1888. Heligel and Hilpath (Hellriegel, H.and H. Wilfarth, 1888 Untersuchungen uber die stickstoff nahrung der gramineen and leguminosen Beilageheft zu der ztschu ver Rubenzuker Industries, Deutschen Reichs, p.234.) By presenting the role, agricultural applicability was revealed.

Root fungus infects roots with specific affinity for a particular host legume and maintains symbiosis with the host.When root fungus is infected with legume roots, it supplies nitrogenous nutrients to plants and obtains the necessary energy from plants. will be. However, mycorrhizal fungi vary not only in the degree of survival, but also in the physiological physiological fixation ability of the host, depending on the cultivation environment of the host, in particular, the soil characteristics. This tendency is even more pronounced when growing alfalfa in acidic soils. R.meliloti, the mycorrhizal fungus R.meliloti, which coexists with alfalfa, is the least resistant to soil acidity among many legumes, and it is very difficult to expect chemical fertilizer replacement effect by mycobacteria-host symbiosis in acid soils.

The hydrogen ion concentration reaction of mycorrhizal bacteria, i.e., the fact that mycobacteria are difficult to survive in acidic soils, is prevalent in Fred and Daveport, 1918. Journal of Agricultural Research. (Washington, DC, 14: 317-336). It has been found in mycorrhizal culture experiments such as Graham and Parker. 1964. Plant Soil (20: 383-394), and this fact is found in Damirgi. 1967.Agronomy J. 59: 10-12, Jensen. 1969. Planteavl. 73: 61-72). In particular, since most of the soil in Korea is acidic, cultivation of alfalfa is necessary to correct the acidity of the soil by using lime even if inoculated with mycorrhizal fungi. However, lime used is not a permanent solution in growing alfalfa, a myocardial crop because of the natural loss caused by rainy season rainfall. Conventional soybean cultivation methods use existing grassland soil as a mycobacterial inoculum, but there are many problems in economical alfalfa grassland composition in acidic soils. This is because mycobacteria distributed in the existing alfalfa cultivation have not only low public nitrogen holding power but also very low density per 1 gram of soil. It is difficult and even more impossible to expect a fixed nitrogen effect.

For peanuts and soybeans, the folk responsiveness of the symbiotic mycobacteria Rhizobium spp. And Rhizobium japonicum to soil reactions is somewhat less than that of Alfalfa mycobacterium Rhizobium meliloti.

Peanut root spp. (Rhizobium spp.) Is expected to have a high inoculation effect because its viable cell count tends to be very low even in the soil where peanuts have been grown for many years, because the existing peanut cultivation area was mainly limited to sandy or sandy soil. do. In addition, when inoculated with mycorrhizal fungi, it is expected that vigorous growth of above-ground plants can be expected even during the harvesting period, and thus, the plant foliage after harvesting has a very high use value as a feed compared to those without the inoculation of mycorrhizal fungi. In addition, Peanut mycorrhizal bacteria have an inoculation effect on red beans, mung beans, and eastern parts belonging to the Cowpea group (Cowpea group) in the taxonomics of legumes has a wide range of applications.

Soybean Rhizobium japonicum is distributed more than alfalfa and peanut root bacteria in arable land in Korea, but it is very difficult to expect the effect of soybean growth with only these native root bacteria because of its low ability to fix nitrogen. In addition, indigenous mycorrhizal bacteria in certain soils are more prone to root infection than soybeans selected from other regions. Rhizobium japonicum's selection is urgently needed.

As such, mycorrhiza found in a particular soil adapts well to its natural location environment, ie the climate and physical and chemical properties of the soil, and forms a symbiotic relationship with the corresponding soybean crop. In order to increase the yield of larvae, a wide range of host-affinity mycobacteria, which are present in domestic soil with a similar soil environment, should be developed as a seed crop inoculation.

The study of commercial use of mycobacterial inoculum began in 1956 in Australia between the University of Sydney and the agricultural authorities, followed by the Burton, J, C.1964. The Rhizobium-Legume Association, in Microbioay and Soil in the 1960s. Fertility, RL 1965. Agronomy J. 57: 379-381. The medium for culturing mycobacteria was Vincent (Jin 1970, A manual for the practical study of root-nodule bacteria). Int. Biol. Program. 15: 54-58.) By YEM medium (1g yeast extract, mannitol 10g K ₂ HPO ₄ 0.5g, MgSO ₄ , 7H ₂ O 0.2g, NaCl 0.2g, FeCl ₃ 6H ₂ O 2mg, distilled water 1L, pH neutral It was developed under the name of) and has been widely used for academic and industrial use.

Strijdom, BW and CCDeschodt. 1976. Carriers of rhizobia and the effect of prior treatment on the survival of rhizobia.p. 151-168.In PS Nutman (ed.), Symbiotic nitrogen fixation in plant.Cambridge University Press, Cambridge) and Brockwell, J. 1977. Application of legume seed inoculants.p. 277-309 In RWF Hardy and AH Gibson (ed), A treatise on dinitrogen fixation.IV.Agronomy and ecology.John wiley & Sons, Inc., New York.) Is a preferred method for inoculating legumes in seed crops grown at 30 ± 2 ° C in YEM medium, such as peat, charcoal and vermiculite. Carrier use of materials has been announced, and Chao, W. L, and M. Alexander 1984. Mineral soil as carriers for rhizobuim inoculants.Appl. Environ. Microbiol. 47 (1): 94-97) We have also confirmed the availability of carriers for mineral soils. Sterilization of carriers for the production of mycobacterial inoculum was carried out using radioscopy (Roughley, R. J and DJ Pulsford. 1982. Production control of legume inoculants. P. 192-209. In JM Vincent (ed.), Nitrogen fixation in legumes.Academic Press, Sydney.) Or autoclaving (P. Somasegaran. 1985. Inoculant pruduction with diluted liquid cultures of Rhizobium spp., Peat, sterlity requirements, storage, and plant effertiveness.Appl.Environ.Microbiol. 50 (2): 398: 405.).

Root fungus produced by the above method is generally inoculated to seed crops by consumers (farmers) by using adhesive materials such as Arabian gum or carbonated gum. However, these materials are not only difficult to purchase farms, but also inconvenient in working with water solutions before use. In addition, the ideal adhesive material should be able to maintain a high number of mycorrhizal fungi when the mycorrhizal powder is added to the seed crops, and until the seed germinates in the soil. However, when gum gum or carbonated gum is used as a sticking agent for mycorrhizal fungi, myobacteria attached to the seeds show a reduced survival rate due to drying during seeding. There is a disadvantage to be used.

Therefore, the present inventors in growing the alfalfa, peanuts, red beans, mung beans, eastern, soybeans and so on crops, to remove the disadvantages of the prior art and to increase the yield to be distributed in the soil of Korea which can achieve the production cost reduction effect As a result of extensive research on the use of new strong nitrogen-fixed mycorrhizal fungi and appropriate adhesive materials in an appropriate manner, the present invention has been completed.

According to the present invention, a new mycorrhizal bacterium with high viability and nitrogen fixability is provided even in barren field soil or farmland.

In addition, according to the present invention, there is provided a myococcal inoculator for oak crops comprising these new mycobacteria alone or in combination.

In addition, according to the present invention, there is provided an adhesive material that can be used to inoculate a myococcal inoculator into a legume crop.

In addition, according to the present invention, there is provided a mycobacterial inoculum kit (kit) consisting of myopia inoculum and adhesive material.

The mycorrhizal fungi according to the present invention were selected after collecting and cultivating about 800 strains of alfalfa (raizobium merotiti), peanut (raizobidium spesis), and soybean (raizobidium japonicum) symbiotic mycobacteria distributed in Korean soil. R.meliloti YCKa 500, YCKa 622, R.spp. YCK15 and R.aponicum YCK 213.

On the other hand, in the present invention, when inoculating the mycorrhizal agent to seeds such as alfalfa, peanut, soybean, peanut oil, soybean oil, mineral oil, etc., which is convenient to use the farmhouse and can improve the mycobacteria viability of the seeded seed. It was used as the adhesive material of the carrier.

In this way, when inoculating mycorrhizal bacteria using oil as an adhesive substance, the number of viable bacteria attached to the seed is not significantly affected even if the seeding of the seeded seed is delayed a little, so that the seeding work is compared with the conventional method using gum gum or carbonated gum. It can have a time margin and can increase the inoculation efficiency of mycobacteria. In addition, there is an advantage, such as reducing the waste of inoculation. In addition, the adhesive material of the oil has the advantage that can be used more conveniently in farms because it can be enclosed in the product packaging during the production process of mycorrhizal fungi.

As a single group of mycorrhizal bacteria, a normal one is used. That is, the powder is made to pass through 200 mesh or 74um sieve by selecting one of dried peat, charcoal, vermiculite, mineral soil, and compost. This is mixed with 100 g of polypropyrene or polyethylene foam, mixed with an appropriate amount of calcium carbonate with a pH of 6.5-7.0 and 10 ml of water, sealed, and sterilized twice continuously for 1 hour at 1kg / 1cm ² steam pressure at 121 ° C. When sterilized and cooled, refrigerated at 4 ℃ and used for inoculation of mycorrhizal fungi.

Root microorganisms with excellent nitrogen fixation ability were adjusted to conventional YEM medium (Yg extract 1g, mannitol 10g, K ₂ HPO ₄ 0.5g, MgSO ₄ 7H ₂ O 0.2g, NaCl 0.2g, FeCl ₃ 6H ₂ O 2mg, distilled water 1ℓ, pH 6.8) ), 50 ml of mycorrhizal culture solution aseptically inoculated into a sterile carrier bag, and the carrier was diffused to 30 ± 2 Incubation for 2 weeks in the dark at ℃ can produce up to 10 ⁹ myococcal inoculators per gram of carrier. The cultured mycorrhizal fungi can be stored at 4 ° C for 5-6 months, and can be used as seed inoculation agents such as alfalfa, peanut, soybeans for cultivation at least 600 pyeong as one bag of mycorrhizal fungi.

In order to obtain mycorrhizal inoculum having excellent soil environment adaptability and affinity, the mycorrhizal fungi of the present invention may be appropriately inoculated in a carrier bag.

The seed inoculum of the soybean crop thus obtained may be diluted with sticky substances such as peanut oil, soybean oil, mineral oil, and the like, and then divided into the soybean crop seeds, and the separated seeds are preferably sown.

In order to make the mycobacterial agent more convenient to use in farms, mycobacterial bacteriostatic agent according to the present invention may be provided in the form of a kit enclosed with the adhesive material in the product packaging of the inoculant during the factory production process.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail based on an Example.

Example 1

Isolation and Identification of Root Fungi with Excellent Nitrogen Fixation

Over 800 strains were isolated from the roots of alfalfa (raizobium merotiti), peanut (raizobium spesis) and soybean (raizobium japonicum) roots in Korean soil. Then, it was determined that these strains express myoclogenesis and nitrogen fixation activity in the sterilized test and the host inoculation test in the pot to determine the mycobacteria.

[Table 1] Composition of YEM medium for separating mycobacteria

pH 6.8 adjustment

Among them, R. meliloti YCKa 500, YCKa 622, R.spp. YCK 15, R. japonicum, YCK 213, etc. The microbiological characteristics thereof were selected as shown in Table 2.

[Table 2] Microbiological Characteristics of Nitrogen Streptococcus

The lysorium merlotti YCKa 500, the lysorium merlotti YCKa 622, the lysorium spheroids YCK 15 and the lysorium japonicum YCK 213 were assigned to the KFCC on May 2, 1991, respectively, to the KFCC. -10725, KFCC-10726, KFCC-10727 and KFCC-10728.

Example 2

Inoculation Effect of Raizobium Merlotti YCKa 500 (KFCC-10725)

After cultivating the lyzobium merloty YCKa 500 of the present invention using a control mycorrhizal fungi on a nitan carrier, inoculating it with alfalfa using peanut oil as a cohesive substance, and then performing aerial nitrogen in the sparse field soil. Fixation and viability were tested and the results are shown in Table 3 below.

[Table 3] Inoculation Effects of Ryzobium Merlotti YCKa 500 on Bare Fields

* Control mycorrhizal ** mycobacteria

As can be seen from Table 3, the mycorrhizal fungi of the present invention are very superior in nitrogen fixation and viability compared to the control mycobacteria.

Example 3

Inoculation Effect of Raizobium Merlotti YCKa 622 (KFCC-10726)

After incubating the Raizobium merlotti YCKa 622 of the present invention on a charcoal carrier using Raizobium merlotti TAL 1372 as a control mycobacteria, inoculating alfalfa using mineral oil as an adhesive, Fixation and viability were tested and the results are shown in Table 4 below.

[Table 4] Inoculation Effect of Rizobium Merlotti YCKa 622 in Farmlands

As can be seen from Table 4, the mycorrhizal fungi of the present invention have a very high nitrogen fixation ability even at the farmland compared to the control mycobacteria, and also allow the plants to grow better, thereby increasing the weight of the farming industry after harvesting, and thus increasing the value of use as a feed. .

Example 4

Inoculation effect of mixed mycobacterial inoculum

After the inoculation of Rhezium Meriloty YCKa 500, YCKa 622 or a mixture thereof to Alfalfa by the method of Example 2 or 3 was tested for nitrogen fixation ability in wild soils and mature soils and the results are shown in Table 5.

[Table 5] Mixed inoculation effects of lyzobium merotiti by soil

Example 5

Inoculation Effect of Raizobium Cipesis YCK 15 (KFCC-20727)

(1) After incubating the Lysodium spesis TAL 1,000 as a control mycorrhizal bacterium on the present invention, after incubating the Lysodium spesis YCK15 on a Nitan carrier, the peanut oil was used as an adhesive to increase the nitrogen fixation capacity and yield. The effects were examined and the results are shown in Table 6.

(2) In comparison with the case of the non-inoculation and conventional inoculation using the lysodium space YCK 15 inoculum of the present invention, the increase effect of peanut was examined, and the results are shown in Table 7.

[Table 6] Inoculation Effects of Ryzobium Spesis YCK 15

* Standard peanut mycorrhizal density of test package: 5.8 × 10 ³ / soil 1g

** control mycorrhizal ***

Table 7 Peanut Enhancement Effect of Rizobium Spesis YCK 15

* Standard peanut root density of test package: 2 × 10 ² / soil 1g

Example 6

Inoculation effect of Raizobium japonicum YCK 213 (KFCC-10728)

After incubating the Razobium japonicum USDA 110 as a control mycorrhizal bacterium, the Razobium japonicum YCK 213 of the present invention was grown on a charcoal carrier, and then inoculated into soybean using soybean oil as an adhesive, followed by nitrogen fixation and root formation. The strength of the steel was evaluated and the results are shown in Table 8.

[Table 8] Nitrogen Fixation and Competitive Force of Ryzobium Japonicum YCK 213

* Control mycobacteria

As can be seen in Table 8, the Ryzobium japonicum YCK 213 of the present invention is superior in the nitrogen fixation force and in particular the muscle formation competitive force compared to the control bacteria. Moreover, since a lot of foliage is obtained after harvesting, it can also be used effectively as a feed.

According to the legumes mycobacterial inoculum of the present invention, the following effects can be expected.

First, it is convenient to use farmhouse, so there is little labor consumption. Second, it can increase the yield of alfalfa, peanut, soybean in any soil environment at low cost. In particular, it is especially high in the fourth place, and it can be expected to stabilize the aerial nitrogen stably during the entire growth period of the crop, so that no fertilizer needs to be added. Fifth, when constructing alfalfa grassland in the strong acid wild field, Sixth, the inoculation of peanut root fungus into red beans, mung beans, eastern part, etc., the yield of these increases, seventh, crop damage is less damage, convenient for agricultural machinery at the time of harvest, eighth, chemical fertilizer Nitrate nitrogen contamination of harvests can be greatly reduced, and ninth, once a mycobacterial agent is used, it is possible to increase fruit crops for years.

Claims (3)

Rhizobium meliloti YCKa 500 (KFCC-10725), Rhizobium meliloti YCKa 622 (KFCC-10726), Rhizobium spp. YCK 15 (KFCC-) isolated from soil 10727) or a culture obtained by mixing one or two or more of the culture medium of Rhizobium japonicum YCK 213 (KFCC-10728) with a carrier, followed by culturing. Mycobacterium inoculum for increase of legumes. A mycorrhizal inoculator kit comprising a mycorrhizal inoculator and a sticky substance for increasing the crop of claim 1. The kit of claim 2, wherein the adhesive material is selected from peanut oil, soybean oil, or mineral oil.