KR100755509B1 - Novel Azospirillum brasilense CW301 with nitrogen fixing ability as well as growth enhancing effect biofertilizer using them and the preparing process thereof - Google Patents

Abstract

A novel strain Azospirillum brasilense CW301 with nitrogen fixing ability as well as growth enhancing effect is provided to transfer the fixed nitrogen to plants and increase nitrogen absorption of the plants, so that the strain is used as a substitute for chemical fertilizer. A strain Azospirillum brasilense CW301(KACC 91224P) has nitrogen fixing ability as well as growth enhancing effect and is isolated from wheat. The biofertilizer contains Azospirillum brasilense CW301(KACC 91224P) and is prepared by culturing Azospirillum brasilense CW301(KACC 91224P). The growth of plants is promoted by soaking seeds of plants in the cultured medium of Azospirillum brasilense CW301(KACC 91224P), wherein the plant is tomato, Chinese cabbage, lettuce, cucumber, red pepper, Welsh onion or crown daisy.

Description

Novel Azospirillum brasilense CW301 with nitrogen fixing ability as well as growth enhancing effect, biofertilizer using them and the preparing process about}

1 is a result showing the increased nitrogenase (nitrogenase) activity in the crops by the novel azospirylbramylene CW301 strain of the present invention.

Figure 2 is a result showing the nitrogen uptake of the crops increased by the novel azospirylbrasilens CW301 strain of the present invention.

Figure 3 is a result showing the growth promoting effect of the crops (tomato, cabbage, lettuce) increased by the novel azospirylbrasilens CW301 strain of the present invention.

Figure 4 is a result showing the growth promoting effect through the seedlings and packaging assay of the Chinese cabbage increased by the novel azospirylbrambrass CW301 strain of the present invention.

The present invention is a plant fixed to, is in more detail the air nitrogen relates to a novel strain azo RY rilrum bra chamber lances biological fertilizer and a method of manufacturing (Azospirillum brasilense) CW301 strain and using the same, which is to promote nitrogen fixation and plant growth effects The present invention relates to azospiryllum brasilens CW301 strain having excellent ability to improve nitrogen absorption of crops through this, as well as a biofertilizer using the same and a method of manufacturing the same.

Soil microorganisms are used in many areas such as crop growth, material circulation and environmental purification. Among these, nitrogen-fixing bacteria that form crops and commensals to fix nitrogen in the air include Rhizobium ( Rhizobium ), which is a microorganism that forms a symbiotic system with legumes. Nitrogenase is fixed in the root nodules to convert air nitrogen into ammonium nitrogen. Ammonium nitrogen produced from the root nodules is transmitted to the crops through the roots of the crops, and the crops increase the nitrogen content. At this time, the carbon source required by root-knot bacteria is directly supplied from crops. Therefore, nitrogen fixation is very important agriculturally, and crops can be grown without nitrogen deficiency even in soil environment where nitrogen supply is insufficient due to nitrogen fixation of root-knot bacteria.

In addition, there is a group of bacteria that supply carbon source by anti-symbiotic system, secretion of crop roots, and fix aerial nitrogen without forming a complete symbiotic system with host crops. Azotobacter genus and azospi rilrum (Azospirillum) in microorganisms, anaerobic bacteria Clostridium (Clostridium) in the conditions in microbial anaerobic bacteria keulrep when Ella (Klebsiella) is typical. Azo RY rilrum (Azospirillum) in the plant growth promoting rhizosphere microorganisms in double fungus; well known as (PGPR Plant Growth Promoting Bacteria) ( Bashan and Holguin, 1997). Azospirylrum microorganisms have been known to form anti-symbiotic systems with wheat, tropical grasses, corn and cereals. In addition, the formation of a biological fertilizer azo RY rilrum (Azospirillum) using a microorganism of the genus and even very advantageous not only nitrogen supply side of the crop promotes crop growth azo RY rilrum (Azospirillum) half symbiotic system and in microbial and crops supply crop nutrients It can play a significant role in increasing efficiency. Therefore, it is very important to increase the agricultural usability to develop a microbial agent that acts as a biofertilizer by separating the bacteria having more excellent nitrogen fixing ability.

A major technical challenge for eco-friendly organic farmers who do not use pesticides and chemicals at all is the poor nutrition of crops. In order to solve this problem, these farmers often buy and use commercially available non-standardized, eco-friendly agricultural materials, or use large quantities of organic fertilizers, which are manufactured in-house, for the purpose of nourishing scarce crops. This increases the type and quantity of input materials indefinitely, adversely affecting the environmental ecosystem as well as the increase in operating costs.

Soil microbial formulations have a variety of effects depending on the purpose of use as a formulation prepared using microorganisms separated from farmland or forest land. Microbial agents consist of active microorganisms, organonutrients for activating microorganisms, and other auxiliaries. The microbial agent has been identified the main factors and components that exhibit the microbial treatment effect, the efficacy should be clearly defined. Although the efficacy may vary depending on the characteristics of the useful microorganisms, it is mainly used for promoting organic decomposition, improving crop productivity, improving crop quality, improving crop disturbance and soil physicochemical properties, improving soil microorganisms, and purifying pollutants. Among them, symbiotic bacteria that directly interact with crops are more obvious in assessing their function than other bacteria because they directly or indirectly affect the physiological mechanisms in the crop.

The nitrogen-fixed microbial fertilizer and its manufacturing method (Korean Patent No. 23999) and soil-treated microorganisms and methods for obtaining the same have been published for the strains that have been isolated and developed as fertilizers (Korea Patent No. 10-2004-7002210). There is a bar. However, the above information focuses on the manufacturing process of inoculating a series of microbial populations into waste to make microbial fertilizers or a process of manufacturing microbial preparations by combining a series of microbial isolates from Hungarian soils with different environments. It is related to the isolation of strains with excellent nitrogen fixation ability, which is purely adaptable to the domestic soil environment, and did not find any difference in the nitrogen supply power of the crop itself.

Accordingly, the present inventors have identified a new azospiryllum brasilens CW301 strain (KACC 91224P) which has the ability to fix the air nitrogen directly from domestic soil, and the present invention is a novel strain to the nitrogen absorption of the crop The present invention has been completed and found to have excellent ability to promote crop growth by promoting.

The present invention has been proposed to solve the problems of the prior art as described above, an object of the present invention is a novel azospirilum brasilens CW301 which is excellent in fixing aerial nitrogen, improving the absorption of crop material and promoting crop growth. To provide a strain. It is another object of the present invention to provide a microbial agent comprising the azospirylbrasilens CW301 strain as an active ingredient. In addition, another object of the present invention to provide a method of using as a biofertilizer to promote the growth of crops by using the Azospirilbrabraens CW301 strain.

In order to achieve the above object, the present invention provides azospirillum brasilense CW301 strain (KACC 91224P) having a nitrogen fixing ability and crop growth promoting effect.

In addition, the present invention provides a biological fertilizer comprising the azospirilbracilens CW301 strain (KACC 91224P).

In addition, the present invention provides a method for producing a biofertilizer by culturing Azospirilbrabraens CW301 strain (KACC 91224P).

In addition, the present invention provides a method for promoting crop growth comprising immersing seed of a crop in a culture medium in which the strain is cultured, and coating the seed.

In the above, the crop includes tomatoes, cabbages, lettuce and vegetable crops such as cucumbers, peppers, leeks, garland chrysanthemum.

Hereinafter, the present invention will be described in more detail.

Azospirilum brassislen strain CW301 of the present invention has an excellent effect to promote crop growth by increasing the nitrogen absorption of the crop by fixing the aerial nitrogen.

The azospirylbramylene CW301 strain of the present invention is a microorganism which is widely distributed in the soil as aerobic, nitrogen-fixing gram-negative bacteria, and forms a symbiotic system with feed crops, grains and non-bean legumes. At the time of infection, the bacteria adhere to the crop roots and are mainly present in the root zone, which is a space within 2 mm from the root surface. The azospiryllum brasilens CW301 strain of the present invention shows a positive reaction to an oxidase reaction, contains poly-beta-hydroxybutyrate particles, and an acetylene-reduction activity (ARA). Indicates. In addition, the strain forms a biofilm in a nitrogen-free semi-solid (Ntrogen-free (NFB) medium).

The azospirylbramylene CW301 strain of the present invention has a nitrogen fixation ability to convert gaseous nitrogen in the air into ammonium form, so that crops can be grown without deficiency even in a nitrogen-deficient condition. Nitrogen fixation ability can be recognized by the nitrogen fixing enzyme (nitrogenase) activity acetylene gas is reduced to ethylene gas by this enzyme, so the activity of nitrogen fixation enzyme was confirmed by measuring the amount reduced from acetylene gas to ethylene gas (Fig. 1). Reference). Strain of the present invention there are dependent on each other names such discrete elsewhere in this capacity; was the superior compared to other strains with a (azo RY rilrum bra chamber lances Azospirillum brasilense) determine excellence ability nitrogenase (see Fig. 1). With this excellent nitrogen fixing ability it was confirmed that to move it to the crop to increase the nitrogen uptake of the crop (see Figure 2).

The present invention can be used as a microbial agent because it improves the nitrogen uptake of the crop as well as promotes the growth of the crop by using the strain of the present invention. The microbial agent may include an azospirylbrasilens CW301 strain as an active ingredient. The microbial preparation according to the present invention may be prepared in the form of a liquid fertilizer and may be used in the form of powdered powder by adding an extender thereto or granulated by formulating it. However, the formulation is not particularly limited. In other words, it can be formulated as a biofertilizer to overcome this in eco-friendly organic farming where the chemical fertilizer supply is limited.

The present invention provides a method for use as a biofertilizer to promote nutrient absorption and growth of crops using azospirylbramylene CW301. Using the culture medium cultured the strain it can be used as it is irrigation in a liquid state, immersed or sprayed on the seed of the crop or coated on the seed.

Crops capable of promoting nitrogen absorption and growth of crops by the method of the present invention may include vegetable crops such as cucumbers, peppers, green onions, garland chrysanthemum, as well as tomatoes, cabbage and lettuce.

Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the contents of the present invention, but the scope of the present invention is not limited to these embodiments.

Example 1 Isolation of Strains

<1-1> Isolation of Strains from Wheat

(1976)의 방법에 의해 균주를 분리하였다.In order to isolate the strains in the root of the wheat root ( Triticum aestivum ), 50-mg / L yeast extract was added using semi-solid malate medium (NFB).

The strain was isolated by the method of (1976).

 <1-2> Identification of Isolated Strains

The nucleotide sequence of the rDNA of the selected nitrogenous bacterium was analyzed according to a conventional method known in the art. As a result, the 16S rDNA base sequence (SEQ ID NO: AY518780) of the nitrogen-fixing bacterium was confirmed to be 99% identical to the base sequence of 16S rDNA of azospiryllum braylene. Physiological biochemical properties of the strains of the present invention are listed in Table 1 below.

Table 1. Biochemical Properties of Nitrogen Streptococcus Strains of the Invention

Strain Host Gram reaction Oxidase reaction BMS badge Use of carbon source Biotin Requirement saccharose glucose Malic acid Azospiril Brasilens CW301 wheat - + pink - - + -

Therefore, the strain was named "Azospirilbrasilens CW301" and it was deposited on March 13, 2006 to the Korea Agricultural and Microbiological Conservation Center, Korea Institute of Agricultural Biotechnology (Accession Number: KACC 91224P).

Example 2 Determination of Nitrogen Fixing Force by Azospiryl Brasilens CW301

In order to determine the efficiency in the nitrogen fixation of the strain of the present invention was examined through the nitrogen fixing enzyme (nitrogenase) activity showing a nitrogen fixing ability. Nitrogen fixase activity was measured by the ARA method (Han and New, 1998) in which the amount of acetylene gas is reduced to ethylene gas. In order to determine the activity of nitrogen fixase by pure strain culture, NFB medium without a nitrogen source was inoculated, inoculated with the strain and incubated for 48 hours at 30˚C, and then the gas phase was acetylene: air: nitrogen at 10: 10: 80 volume ratio. After substituting the mixed gas with decreasing oxygen partial pressure, samples were taken at intervals of 24 hours to measure the amount of ethylene generated through gas chromatography. In addition, in order to investigate the activity of nitrogen fixase by strain inoculation when the strain formed anti-symbiotic system with the crop, wheat was used as host crop and cultured in NFB medium without nitrogen added with 0.05% yeast extract for 8 hours or more. Two weeks after inoculation with one of the above strains, the nitrogen-enzyme activity was examined using the ARA method. Control group, azo RY rilrum bridal chamber Lawrence (Azospirillum brasilense) Sp7 (Tarrand et al., 1978) and of the same species name separated from Australia azo RY rilrum lipoic perum (Azospirillum lipoferum) was used for 687 inhabit the roots around the various plants separating and various azo RY rilrum bra chamber lances (Azospirillum brasilense) strains were compared to the nitrogenase enzyme activity. As a result, as shown in FIG. 1, when the strain Azospirillum brasilense CW301 of the present invention formed a semi-symbiotic system with the host crop, it was confirmed that the activity of nitrogen fixing enzymes (nitrogenase) was much higher. .

Example 3 Crop Nitrogen Absorption Enhancement Effect by Azospirylbrasilens CW301

In order to find out how the new strain of the present invention affects the nitrogen uptake of crops, the new strain of the present invention was cultured in TSB medium for 8 hours or longer, and then inoculated into cabbage. Chinese cabbage seeds were sterilized in 70% ethanol for 1 minute and 1% sodium hypochlorite solution for 20 minutes, washed with distilled water 6 times, and soaked in the culture solution of the strain for 2 hours. The control group was used by soaking in distilled water in the same manner. Topical soil was used Vermiculite and 500 times the amount of commercial nutrient solution (Mulpure No. 1) was used (300ml, 3 times / week). After 40 days of sowing, the cabbage seedlings were harvested, dried at 60 ° C. for 3 days, crushed, decomposed by wet decomposition using sulfuric acid-chloric acid, and the total nitrogen content of the crop was measured by Kjeldahl (Kjedahl). As a result, as shown in Figure 2 it was confirmed that the nitrogen content of the crop is increased in the crop body in the Chinese cabbage treated with the new strain CW301 of the present invention improved the nitrogen absorption of the crop.

Example 4 Crop Growth Enhancement Effect by Azospirillum Brasilens CW301

<4-1> Tomato, Chinese Cabbage, Lettuce Growth Promoting Effect by Azospirillum Brasilens CW301

In order to examine the effect of the new strain of the present invention on the growth of crops, three crops of tomato, cabbage and lettuce were examined. Azospirilbrasillan CW301 was incubated in TSB medium for 8 hours as in Example 3, and the seed of each crop was soaked in the culture solution for 2 hours and then sown. Seeds were sterilized with 70% ethanol and 1% sodium hypochlorite before strain inoculation and washed with distilled water. The control group was soaked in distilled water in the same manner. As the topsoil, sterilized vermiculite was used, and nutrient solution was used as a commercial nutrient solution (Mulpure No. 1) 500 times (300 ml, 3 times / week). After 40 days of sowing, the growth of each crop was observed. As a result, as shown in Figure 3, the crops inoculated with the new strain CW301 showed significantly higher growth of both tomatoes, cabbage, and lettuce.

<4-2> Effect of Chinese Cabbage Growth on Packaging Assay by Azospirillum Brasilens CW301

To examine the effects of the new strain of the present invention on the growth of crops more clearly, pavement test was performed using cabbage seedlings. Filling 1 / 2000a Wagner pot with 17kg of soil (main container, specification quality) was treated by mixing rice bran into the soil at a depth of 10cm in the amount of 150kg / 10a. Chinese cabbage was filled with commercial topsoil in a 50-hole plug pot, seeded for 21 days, and then packaged. The method of inoculating new strains and disinfecting seeds before inoculation was carried out in the same manner as in Example 4-1. The seed culture was supplemented with a pipette (100 µl / plant) even when seeded together with seed immersion. As a result, as shown in Fig. 4, even after seeding, not only in seedling state, but also in the cabbage inoculated with the strain Azospirilum brasilens CW301 of the present invention, it was confirmed that the growth promoting effect was significantly higher.

As described above, the novel azospirylbrasilens CW301 strain of the present invention has an excellent ability to fix aerial nitrogen, and has an excellent effect of promoting crop growth by improving nitrogen absorption of crops. Therefore, the novel strain of the present invention can be used to increase the yield and quality of crops by supplying nitrogen to crops in place of chemical fertilizers in eco-friendly organic farming where the use of fertilizers is limited.

In addition, the present invention provides a microbial fertilizer having excellent nitrogen fixation power by using the characteristics of nitrogen-fixed bacteria to fix the air nitrogen as a biological fertilizer not only to supply the crops nutrients due to the reduced use of chemical fertilizers, but also contribute to the preservation of environmental ecosystem It can be actively used for production.

Attach sequence list electronic file

Claims (6)

Azo RY rilrum bra chamber lances (Azospirillum with nitrogen fixation and promote plant growth effect brasilense ) CW301 strain (KACC 91224P).  Biological fertilizer containing the azospirilbrasillan CW301 strain (KACC 91224P) of claim 1. A method for producing a biofertilizer by culturing azospirilbracil CW301 strain (KACC 91224P). Method of promoting the growth of crops by immersing the seeds of the crop in the culture medium cultured strain of claim 1. The method of claim 4, wherein the crop is a vegetable crop. The method of claim 5, wherein the crop is any one or more selected from tomatoes, cabbages, lettuce, cucumbers, peppers, green onions, garland chrysanthemum.

Images