KR20170049454A - Lactobacillus plantarum DGK-17 strain promoting seed germination of plant and uses thereof - Google Patents

Abstract

The present invention relates to: a lactobacillus plantarum DGK-17 strain for promoting seed germination of a plant; a microorganism medicine and an organism fertilizer for promoting seed germination of a plant comprising the strain, a culture medium thereof, and a concentrated liquid of the culture medium or dried products thereof as active ingredients; a method for producing the organism fertilizer comprising a step for culturing the strain; and a method for promoting seed germination of the plant comprising a step for treating the strain with seed of the plant. The lactobacillus plantarum DGK-17 strain of the present invention can promote seed germination of the plant, thereby being expected to contribute to a production increase of the plant by using the same.

Description

Lactobacillus plantarum DGK-17 strain promoting seed germination of plants and use thereof Lactobacillus plantarum DGK-17 strain promoting seed germination of plant and uses thereof

The present invention relates to a lactobacillus plantarum DGK-17 strain which promotes seed germination of a plant and a use thereof. More particularly, the present invention relates to a lactobacillus plant strain DGK-17 which promotes seed germination of a plant and more particularly to a lactobacillus strain DGK-17 having an effect of increasing seed germination rate of peanut, Lactobacillus plantarum DGK-17 strain.

Peanuts ( Arachis hypogaea ) belonging to the Fabaceae are important crops globally due to their high nutritional value and oil content. Peanuts are a major oilseed crop on the planet. Peanuts occupy a unique position among fatty seeds because they can be used in a variety of ways and can be consumed directly. More than half of world peanut production is shredded for oil release. Peanut cake, a by-product of the oil industry, is mainly used as a livestock feed supplement. Many health benefits associated with peanut consumption have been reported, including weight control, prevention of cardiovascular disease, protection against Alzheimer's, and cancer suppression. These benefits include the fact that peanuts contain low levels of saturated and trans-fatty acids, as well as mono-unsaturated fatty acids, poly-unsaturated fatty acids, and vitamin E, folate, , Fiber, and health-promoting phytochemicals resveratrol and other phenolic compounds. Peanut seeds contain 40-50% oil and 27-29% protein and provide a good source of protein compared to many other nuts.

Barley ( Hordeum vulgare L.), a member of the Poaceae grass, is a major grain and is considered to be the next most important crop after wheat, corn and rice among the top ten crops in the world. Globally, 85% of barley production is used for feeding cattle, pigs, and poultry. The second most popular use of barley is malt, which is mainly used in beer, and is used as a spice in distillers, mackerel and various foods. Barley is a great source of dietary fiber, protein and complex carbohydrates, and is a good report of certain vitamins and minerals. However, the composition is markedly different between different environments and cultivars. Carbohydrates, including starch, sugar and non-starch polysaccharides, make up about 80% of the barley kernel. The majority of carbohydrates are present in the form of starch (60%). As a whole, the non-polar polysaccharides of barley, termed dietary fiber, have many health benefits.

Germination is defined as the development and development of a major structure from a seed embryo showing the ability to produce normal plants under favorable conditions. Seeds require specific conditions for normal germination, the most important being substrata, moisture, temperature and light. Apart from these basic requirements, many other physical, chemical and biological factors have an important influence on seed germination.

The abovementioned peanuts and barley are major crops with high economic value and require faster and higher germination efficiency to achieve their required production.

In the present invention, when a synthetic microorganism (beneficial microbe (BM)) Lactobacillus plantarum DGK-17 is mixed in a synthetic medium and placed in a specially prepared perforated germination box, And the effect of beneficial microorganisms on seed germination of ginseng and ice plant.

Korean Patent Laid-Open No. 2009-0005917 discloses a composition for inhibiting seed germination using Pseudomonas sp. And a method for producing the same, and Korean Patent No. 0991298 discloses a method for producing seeds using Arthrobacter zoysiae Which is capable of inhibiting the germination and growth of the plant, is disclosed. However, Lactobacillus plantarum DGK-17 strain which promotes seed germination of the plant of the present invention and its use has not been described.

The present invention has been made in view of the above-described needs. The present inventors have found that a lactobacillus plantarum DGK-17 strain ( Lactobacillus plantarum DGK-17), which is a useful microorganism, is diluted and mixed in a medium and then mixed with a peanut, The seed germination rate of the peanut, barley, ginseng and ice plant was increased in the experimental group treated with diluted Lactobacillus plantarum DGK-17 strain compared with the control without the strain, , Thereby completing the present invention.

In order to solve the above problems, the present invention provides Lactobacillus plantarum DGK-17 strain having plant seed germination promoting effect.

The present invention also provides a microorganism preparation for promoting seed germination of a plant comprising the strain, a culture thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.

The present invention also provides a biosimilar for promoting seed germination of a plant comprising the strain, a culture thereof, a concentrate of the culture broth or a dried product thereof as an active ingredient.

In addition, the present invention provides a method for producing a biological fertilizer comprising culturing the strain.

The present invention also provides a method for promoting seed germination of a plant comprising the step of treating the strain with the seed of the plant.

The Lactobacillus plantarum DGK-17 strain of the present invention was found to have an effect of increasing the seed germination rate of plants. Therefore, it is expected that the microorganism preparation containing the Lactobacillus plantarum DGK-17 strain can promote seed germination of the plant, and if used, it can contribute to the increase of the plant production.

Figure 1 shows the results of the analysis of the effect of germination of peanut on Lactobacillus plantarum DGK-17 treatment. 500X BM, Lactobacillus plantarum DGK-17 500-fold dilution; 200X BM, Lactobacillus plantarum DGK-17 200-fold dilution.
FIG. 2 shows the results of the analysis of the effect of germination of barley seeds on Lactobacillus plantarum DGK-17 treatment.
FIG. 3 shows the results of the analysis of the germination effect of ginseng seeds on Lactobacillus plantarum DGK-17 treatment.
4 shows the results of the analysis of the effect of the germination rate of the ice plant seeds on Lactobacillus plantarum DGK-17 treatment.

In order to accomplish the object of the present invention, the present invention provides Lactobacillus plantarum DGK-17 strain having plant seed germination promoting effect.

In the strain according to one embodiment of the present invention, the Lactobacillus plantarum DGK-17 strain may be KACC81028BP, but is not limited thereto.

The Lactobacillus plantarum DGK-17 strain of the present invention was isolated from kimchi, a traditional Korean fermented food. The 16S rRNA sequence analysis was followed by UPGMA and MEGA 4 programs. It is confirmed that it is 100% with Room_WCFS1 (NR_075041.1). The above strain was deposited at the Institute of Agricultural Biotechnology on Oct. 11, 2016 (Accession No .: KACC81028BP).

In a strain according to one embodiment of the present invention, the plant is selected from the group consisting of peanut, ice plant, soybean, mung bean, kidney bean, pea, potato, eggplant, tobacco, pepper, tomato, burdock, lettuce, bellflower, spinach, , Celery, carrot, buttercup, parsley, cabbage, cabbage, gab, watermelon, melon, cucumber, pumpkin, pak, strawberry or barley, ginseng, rice, wheat, rye, corn, sugarcane, oats and onions And preferably a peanut, an ice plant, barley or ginseng, but is not limited thereto.

In addition, the present invention provides a microorganism preparation for promoting seed germination of a plant comprising the strain of the present invention, a culture thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.

The present invention also provides a biological fertilizer for promoting seed germination of a plant comprising the strain of the present invention, the culture liquid thereof, the concentrated liquid of the culture liquid or the dried material thereof as an effective ingredient.

In the present invention, the term "cultured product" means that a specific microorganism is cultured in a culture medium or a culture solution, and the culture product may or may not include a specific microorganism, but preferably includes a microorganism. The culture may be, but is not limited to, a liquid or solid formulation.

In the present invention, the microorganism preparation may be used as a single species of the Lactobacillus plantarum DGK-17 strain of the present invention, or may be a mixture of two or more microorganisms. When two or more kinds of microorganisms are mixed, Lactobacillus plantarum DGK-17 and microorganisms capable of promoting seed germination of plants may be included.

In the present invention, the microbial agent may be in the form of solid or liquid, but the formulation is not particularly limited. In other words, it can be formulated as biological fertilizer to overcome this in eco - friendly organic agriculture with limited supply of chemical fertilizer.

In the case of the solid microorganism preparation, the microorganism may be attached to the carrier and then dried to a moisture content of 0.1 to 10% by weight, and beads may be produced or pulverized into a powder form. When a microorganism preparation is used in a solid form, the manufacturing process is simple, the production cost is low, and the preservation property is good.

The carrier may be at least one selected from the group consisting of powdery clays, activated carbon, coke, volcanic ash and soft materials. Examples of the clay include zeolite, vermiculite, diatomaceous earth, kaolin, onionite, feldspar, And talc may be used, but the present invention is not limited thereto.

An excipient may be added to the microbial formulation in solid form. In the case of the above excipients, amino acids, vitamin C, vitamin E, chitosan and glucose may be used singly or in combination of two or more, but the present invention is not limited thereto.

In the case of the microorganism preparation in liquid form, it may be prepared by mixing the culture of the microorganism and mixing glucose or glycerin to stabilize the microorganism. When a microbial agent is used in liquid form, it is more active and easier to handle than a powdered product.

The present invention also provides a method for producing a biological fertilizer comprising culturing a strain of the present invention. Any of the methods known in the art can be used for the culture method of the strain and the method for producing the biological fertilizer, and the method is not particularly limited.

The present invention also provides a method for promoting seed germination of a plant, which comprises treating the seed of the plant with the strain of the present invention. As a method for promoting seed germination of the plant, the culture broth in which the Lactobacillus plantarum DGK-17 strain of the present invention is cultured and the microorganism preparation using the strain can be carried out by immersing the seed in the seed, or spraying or spraying the seed. In the case of immersion, seeds can be immersed in the culture medium and the microbial agent.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Isolation and Identification of Strain

Lactobacillus plantarum DGK-17 was isolated from kimchi, a traditional Korean fermented food purchased from a local restaurant. After 16S rRNA sequencing, UPGMA method and MEGA 4 program were used to prepare the phylogenetic tree. As a result, the Lactobacillus plantarum DGK-17 strain of the present invention showed 100% agreement with Lactobacillus plantarum_WCFS1 (NR_075041.1), and Lactobacillus plantarum DGK-17 ( Lactobacillus plantarum DGK-17), and the strain was deposited at the Institute of Agricultural Biotechnology on Oct. 11, 2016 (Accession No .: KACC81028BP).

2. Seed Cleaning

The seeds of the peanut, barley, ginseng and ice plant plants used in the invention were sterilized by dipping in 0.01% mercuric chloride (HgCl ₂ ) solution for 5 minutes, and washed twice with distilled water. Finally, the surface-sterilized seeds were immersed in water for 30 minutes.

3. Seed germination medium

The seed germination medium used may be selected from the group consisting of calcium nitrate tetrahydrate, potassium nitrate, nitrate, iron chelate, magnesium sulfate heptahydrate, magnesium nitrate hexahydrate, ammonium phosphate monoborate, boric acid, manganese sulfate monohydrate, zinc sulfate heptahydrate, After mixing the ribbed sodium bicarbonate, the pH was adjusted to 7.4 and stored at room temperature until further use.

4. Preparation of useful microorganisms

Lactobacillus plantarum DGK-17, a useful microorganism of the present invention, was cultured and maintained in a 2% malt extract liquid medium. To determine the effect of the useful microorganisms on seed germination, useful microorganisms were diluted 200 and 500 times, respectively, in the seed germination medium.

5. Germination test

The germination test was carried out using a specially designed perforated germination box (9 x 9 cm) filled with seed germination medium on a constantly watered base. Seed germination tests were performed in three different groups. Group I was seeds sprayed onto a tray that was just germinated but served as a control, Group II and Group III seeds were sprayed onto trays supplied with germination media containing diluted microbes at 200 and 500 times, respectively . Seed germination was carried out at a temperature of 25 캜 under the condition that the light was shielded from the germination box with the lid.

5.1 Germination percentage

Seed germination was measured daily in each group and the final germination rate of each group was calculated on the last day using the following equation:

5.2 Germination index

The strength of the seeds was determined by measuring germination index values, and the germination index was calculated according to the following equation:

5.3 Mean germination time (MGT)

The average germination time of the seeds was calculated according to the following equation:

Where n represents the number of seeds germinated on the date, and D represents the date calculated from the start date of the experiment.

5.4 50% Germination time 50%, T ₅₀ )

The time taken for germination of 50% seeds (T ₅₀ ) was calculated according to the following equation:

Where N is the final number of germinated seeds, and ni and nj represent the number of accumulated germinated seeds of adjacent seeds that have been harvested at times ti and tj, respectively. (when ni < N / 2 < nj)

5.5 Plant growth measurement

Plant growth was measured to reflect the yield of live weight, dry weight, fresh weight and dry weight, root length, stem length and number of leaves. All of the above parameters were measured after the last cultivation date and the seedlings were dried in a hot air oven at 70 캜 for 24 hours before determining the dry weight.

Example 1. Analysis of the effect of germination rate of peanut and barley seeds on Lactobacillus plantarum DGK-17 treatment

The seed germination rate over time is a more important variable than any other for assessing the effect of the useful microorganism Lactobacillus plantarum DGK-17 treatment. The results of the germination test for peanut seeds showed that the group treated with 200-fold diluted useful microorganisms (63.6 ± 4.3%) was the group treated with 500-fold diluted useful microorganisms (59.1 ± 4.1%) and the control group without microorganisms ± 3.4%) (Table 1).

Analysis of seed germination parameters of peanut treated with Lactobacillus plantarum DGK-17 Control 500-fold dilution treatment group 200-fold diluted treatment group Germination rate (%) 54.5 ± 3.4 59.1 ± 4.1 64.6 ± 4.3 Germination index (GI) 2.16 ± 0.21 2.70 + - 0.12 3.50 + - 0.34 Average germination time (date) 5.33 + - 0.41 5.3 ± 0.61 4.57 + - 0.34 50% germination time (date) 4.5 ± 0.14 4.4 ± 0.11 3.0 ± 0.14 Fresh weight of whole plant (g) / pot 414.9 ± 53.9 434.3 + - 46.8 505.7 ± 51.6 Fresh weight gain (%) 100.0 104.6 122.9 Dry weight (g) of whole plant / pot 208.6 ± 34.7 221.1 ± 31.2 259.8 ± 36.3 Dry weight yield (%) 100.0 105.9 124.5 Root length (cm) 3.8 ± 1.8 4.1 ± 1.4 5.9 ± 1.2 Stem length (cm) 3.01 + - 0.81 3.20 ± 1.1 3.80 ± 1.3 Average number of leaves 10.5 + 4.1 11.8 ± 1.8 13.8 ± 3.1

The germination of peanut seeds appeared within the first 3 days in all groups, reaching a maximum at 7 days and no increase in seed germination after 7 days. The germination rate on the third day of the group treated with the 200 - fold diluted useful microorganism was 31.8%, and the group treated with the 500 - fold diluted useful microorganism and the control group showed 13.64% and 13.64%, respectively (FIG.

Similar results were observed in the germination studies using barley seeds, showing that the 200-fold dilution group of useful microorganisms was superior to the control and 500-fold dilution group in germination rates (Table 2 and FIG. 2). A high level of seed germination of peanuts and barley in the presence of 200-fold dilution useful microorganisms suggests that seed germination of useful microorganisms is effective. The intensity of seed germination in the other treatment groups was determined by calculating the germination index (GI) value. In the peanut and barley seed germination tests, seed germination in the presence of a 200-fold diluted useful microorganism was found to be higher in the 500- , Suggesting the role of a 200 - fold diluted useful microorganism in the potency of seeds. The average germination time (MGT) and 50% germination time (T ₅₀ ) of peanuts and barley were determined in all groups.

Analysis of Seed Germination Parameters of Barley by Lactobacillus plantarum DGK-17 Treatment Control 500-fold dilution group 200 times dilution group Germination rate (%) 28.6 ± 2.4 30 ± 2.1 32.9 ± 1.3 Germination index (GI) 6.3 ± 0.28 7.2 ± 0.31 8.2 ± 0.29 Average germination time (date) 3.65 0.41 3.33 ± 0.32 3.21 ± 0.37 50% germination time (date) 2.83 ± 0.21 2.52 + 0.15 2.35 ± 0.18 Fresh weight of whole plant (g) / pot 5.90 + - 0.63 7.83 + - 0.33 9.11 ± 0.54 Fresh weight gain (%) 100.0 132.7 154.4 Dry weight (g) of whole plant / pot 0.63 + - 0.05 0.72 + 0.07 0.93 + - 0.05 Dry weight yield (%) 100.0 116.0 149.3 Root length (mm) 47.0 7.7 53.5 ± 6.3 65.2 ± 14.1 Stem Length (mm) 36.3 ± 3.1 38.2 ± 7.2 42.5 ± 5.1

The results of the study showed that the average germination time was 500 times higher than that of control group and 500 times useful microbial treatment in peanut and barley group using 200 times diluted useful microorganism. A similar trend was observed at 50% germination time. Based on all the above results on the parameters of seed germination, we propose the usefulness of the 200 - fold diluted useful microbial treatment and its role as seed germination amplifiers in useful microorganisms as a means of seed germination in later studies and germination for dormant seeds And that it can be used for promotion.

Example 2. Analysis of germination rate of ginseng and ice plant seeds treated with Lactobacillus plantarum DGK-17

In addition to the experiments using peanut and barley seeds, the germination efficiency of ginseng ( Panax ginseng CA Mey) and ice plant ( Mesembryanthemum crystallinum ) was further verified by treatment with Lactobacillus plantarum DGK-17, a useful microorganism.

The prepared ginseng seeds were sterilized in the same manner as barley. For the germination and plant growth test, a control (nutrient medium) not treated with microorganisms, a group treated with 500-fold diluted microorganisms, and a group treated with 200-fold diluted microorganisms were classified. Germination experiments of ginseng seeds were carried out at a low temperature of about 4 ℃ for 2 weeks. Fifteen sterilized seeds were placed on a tray (9 x 9 cm) inside a plant growth box, and the medium of each condition was filled. After germination was completed, the plant grower was moved to room temperature, and the lid was opened. Then, the plant was cultured for 26 days to further photosynthesis.

The results of the germination test for ginseng seeds showed that the group treated with 200 times diluted microorganism (60 ± 3.1%) was significantly higher than the group treated with 500 × diluted microorganism (40 ± 2.5%) and the control without microorganism (20 ± 1.4 %) (Table 3 and Fig. 3).

Analysis of seed germination parameters of ginseng treated with Lactobacillus plantarum DGK-17 Control 500-fold dilution group 200 times dilution group Germination rate (%) 20 ± 1.4 40 ± 2.5 60 ± 3.1 Germination index (GI) 0.4 ± 0.05 0.6 ± 0.03 1.2 ± 0.07 Fresh weight of whole plant (g) / pot 180 ± 9 360 ± 16 570 ± 19 Fresh weight gain (%) 100 200 316 Dry weight (g) of whole plant / pot 54 ± 4 112 ± 9 183 ± 12 Dry weight yield (%) 100 207 338.8 Root length (mm) 9.3 ± 0.7 10 ± 0.7 10.9 ± 0.5 Stem Length (mm) 12 ± 1.1 46 ± 3.2 54.25 + - 4.7 Number of leaves 2 2 2

Germination experiments on ice plant seeds were carried out in the same manner as other seeds. The experimental groups were divided into three groups: control (nutrient medium) without treatment of microorganisms, group treated with 500-fold diluted microorganism, and group treated with 200-fold diluted microorganism. Seed germination and growth were observed at room temperature Day.

The results of the germination test on ice plant seeds also showed that the group treated with 200-fold diluted microorganism (82.5 ± 4.2%) was the group treated with 500-fold diluted microorganism (54 ± 2.2%) and the control without microorganism (52.5 ± 3.2%) (Table 4 and Fig. 4).

Analysis of seed germination parameters of ice plant according to Lactobacillus plantarum DGK-17 treatment Control 500-fold dilution group 200 times dilution group Germination rate (%) 52.5 ± 3.2 54 ± 2.2 82.5 ± 4.2 Germination index (GI) 6.5 ± 0.47 6.7 ± 0.34 11 ± 0.92 Average germination time (date) 4.8 ± 0.25 4.7 ± 0.27 3.5 ± 0.21 Fresh weight of whole plant (g) / pot 52.5 ± 3.2 56.2 ± 3.6 108.9 ± 5.1 Fresh weight gain (%) 100 104 201 Dry weight (g) of whole plant / pot 5.88 + 0.08 7.13 ± 0.08 19.8 ± 0.02 Dry weight yield (%) 100 121 336 Root length (mm) 8.4 ± 1.1 9.3 ± 1.4 15 ± 3.9 Stem Length (mm) 10.5 ± 1.4 11.2 ± 1.9 17.1 + 1.7 Number of leaves 2 2 2

It was confirmed from the above results that the Lactobacillus plantarum DGK-17 strain of the present invention affects various variables including the germination rate in relation to the seed germination of the plant, It is expected that it could be used for promotion of germination of dormant seeds as an enhancing means.

Agricultural Biotechnology Research Institute KACC81028BP 20161011

Claims (6)

Lactobacillus plantarum DGK-17 strain (KACC81028BP) having plant seed germination promoting activity. The Lactobacillus plantarum DGK-17 strain according to claim 1, wherein the plant is a peanut, barley, ginseng or ice plant. A microbial preparation for promoting seed germination of a plant comprising the strain of claim 1 or 2, a culture thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient. A biological fertilizer for promoting seed germination of a plant comprising the strain of claim 1 or 2, a culture thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient. A method for producing a biological fertilizer comprising culturing the strain of claim 1 or 2. A method for promoting seed germination of a plant comprising the step of treating the strain of claim 1 or 2 to the seed of the plant.

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