KR100376800B1 - Novel Serratia marscens mutants having activities for plant growth promotion and damping-off control - Google Patents

Abstract

The present invention relates to novel Serratia marscens mutant strains N4-5 and N1-8 having plant growth promoting and control of stocking diseases.

Description

Novel Serratia marscens mutants having activities for plant growth promotion and damping-off control

The present invention relates to a novel Seracia marsense mutant strain having plant growth promoting and control of stock diseases.

Receiving diseases occur in almost all crops, especially young seedlings, and cause severe damage to peppers, tomatoes, eggplants, watermelons and cabbages. It is a disease that occurs a lot in germination or seedlings. When this disease occurs, the seed sown may not germinate, and even when germinated, it does not come out on the ground and dies. Therefore, when the disease is severe, the damage that causes the lack of seedlings is a big disease that occurs all over the country. The symptom of the wearing disease begins when the cotyledon develops by invading the branch of the stem, and the cotyledon develops into dark brown with softening of the branch of the branch, and becomes easily broken after being cut off. Die. When it occurs at the time of 2 ~ 3 sheets of main leaf, it first wither in the day and recover at night, and then fall down from the branch of the jijo. The basal part softens while showing the water immersion phase, and the stem becomes thinner and expands gradually under the high humidity conditions to form white mold. In the seedling stage, the seedlings may be destroyed by pathogens such as Rhizoctonia , mycobacterium, and late blight, in addition to Pythium . In particular, it is difficult to distinguish the symptom between pisium and lysatonia, but it is characterized by dark brown.

Receiving pathogens are a type of filamentous fungi and belong to incomplete fungi and produce mycelia and mycelia on the surface of diseased tissues. Mycelium is brown, thick, branched at right angles, and branched. The bacterium is brown indefinite and not uniform in size. Breeding mainly by hyphae, it is possible to form gall bladder during hot summer. Because it is multiple, it invades and invades various kinds of vegetables. The growth temperature is 13 ~ 15 ℃, optimal 24 ℃, and 40 ~ 42 ℃, and the soil acidity necessary for development is pH 3.0 ~ 9.5, which is good in weakly acidic soil. Develop.

Various methods are known for controlling wear diseases. For example, seedling soils use unpolluted soil, such as mountain soil or subsoil, as far as possible. If soil is concerned about contamination, chemically disinfect with chloropicrine, methyl bromide and pyrohydrating agent two to three weeks before use, and after sufficient gas removal for more than one week, use steam sterilization. It is used later as topsoil. It is known that seed can be reduced to some extent by sowing the seed with homai (gold hydrating agent) or immersion with benrate tee (benoram hydrating agent, big hydrating hydrous) before sowing. Another option is to give eggplant hydration directly to the soil just before sowing.

As an alternative, natural antagonistic bacteria can be used to combat incoming diseases. Yoon SH, J. Microbiol. And Biotechnol., Vol. 9, Iss 3, pp. 352-357, 1999; And Burkhead KD, et al., Biochem. Vol. 30, Iss 5, pp. 665-667, 1998 discloses that several antagonistic strains belonging to the Enterobacter species can be used as fungal phytoprotectants against the incoming pathogen Pythium ultimum . Howell, CR, et al., Phytopath, 70: 712-715, 1980 discloses that Pseudomonas fluorescens can be used to suppress incoming diseases caused by P. pellucida during cotton sowing. Doing. U.S. Pat.Nos. 5,403,583 and 6,291,426 disclose the use of Bacillus spp. To control plant diseases caused by Lizatonia and Pisium bacteria. U.S. Patent No. 6,110,726 discloses the use of actinomycetes as a plant disease control agent by pisium bacteria.

Although some microorganisms have been successfully used to control crop diseases with crops, there is still a need to improve antagonistic activity against stock pathogens. An object of the present invention is the development of a new strain that exhibits excellent antagonistic activity against the pathogens of crops.

The present invention provides a novel Seracia marsense mutant strain N4-5 having plant growth promoting and control of stock diseases.

The present invention also provides a novel Seracia marsense mutant strain N1-8 having plant growth promoting and control of stocking disease.

1 is a result of analyzing the cell fatty acid type and distribution of Ceracia marsense N4-5 according to the present invention by MIDI.

2 is a result of analyzing the cell fatty acid type and distribution pattern of Ceracia marsense N1-8 according to the present invention by MIDI.

3 is a result of measuring the stem length of the cucumber plants grown by sowing after immersing the cucumber seeds in strain Seracia marsense N4-5 according to the present invention.

4 is a result of measuring the stem thickness of the cucumber plants grown by sowing after immersing the cucumber seeds in strain Seracia marsense N4-5 according to the present invention.

Figure 5 is a result of measuring the leaf length of the cucumber plants grown by sowing after immersing the cucumber seeds with strain Ceracia marsense N4-5 according to the present invention.

Figure 6 is a result of measuring the side width of the cucumber plants grown by sowing after immersing the cucumber seeds with strain Ceracia marsense N4-5 according to the present invention.

The present invention relates to a strain of Serritia marscens having excellent plant growth promoting and control of stocking diseases . These strains were isolated from the roots of cucumber seedlings by selecting species that dissolve chitin in plate media. The strains of the present invention are classified by Bergey's Manual of Determinative Bacteriology and Systematic Bacteriology and identified by microbial identification system (MIDI; Microbial ID, Inc., Newark, Delaware, USA) using the type and distribution of cellular fatty acids. New variant strains belonging to the sense species were identified.

The novel variant strains isolated and identified according to the present invention are named Cerasia marsense N4-5 and N1-8, respectively, and the accession numbers on October 11, 2002 to the Korean Culture Center of Microorganisms under the Budapest Convention, respectively. Deposited as KCCM 10434 and KCCM 10433.

The bacteriological properties of Ceracia marsense N4-5 and N1-8 according to the present invention are as described in Table 1 below.

Property data Morphological Characteristics on Medium Pink red round colony formation (cold red color becomes colder) Microscopic characteristics Bacillus Gram reaction voice Optimum growth temperature 30 ℃ Growth temperature 10 to 42 ° C Growth medium Nutrient Agar, Luria-Bertani Badge Culture method Liquid Media-Shaking or Solid Media-Politics How to store At -80 ° C in 20% glycerol stock

The invention will be more specifically illustrated by the following examples. However, these examples are merely embodiments of the present invention and do not limit the scope of the present invention.

( Example 1)

Isolation of Microorganisms

Healthy roots of cucumber seedlings were placed in sterile water and treated with ultrasound for 5 minutes, and then dilution plating was performed on nutrient agar plates to dissolve chitin. These bacteria were shaken and cultured at a temperature of 30 ° C. after inoculation into nutrient agar medium according to a conventional method.

( Example 2)

Analysis of Cell Fatty Acid Types and Distribution Patterns of Microorganisms

The cell fatty acid types and distribution patterns of the strains isolated from Example 1 were analyzed. Methyl esterification of fatty acids and methyl phenyl silicone fused silica capillary column on gas chromatograph (Hewlett-Packard Model 5890A) by standard test method according to microbial identification system of MIDI (Microbial ID, Inc., Newark, Delaware) 102, 25 mx 0.2 mm), and analyzed the distribution patterns of fatty acids with Chromatopac CR 4A data analyzer.

1 and 2 show the analysis results. Strains isolated from these results were classified as strains of Cerasia marsense and named N4-5 and N1-8, respectively.

( Example 3)

Germination rate measurement experiment by microorganism

Cucumber seeds (variety: Cucumis sativus L. Jin Hung Shinpoongcha) were soaked in 0.5% methylcellulose (control) or soaked in 0.5% methylcellulose added with a concentration of 10 ⁸ cfu / ml for 5 minutes. The top soil (enpico horticulture, Co., Ltd. farm) was put in a pot 6 cm wide, 6 cm depth to plant two to three cucumber seeds per pot to measure the germination rate for a total of 50 cucumber seeds after 4 days. Growth conditions were irradiated with artificial light (white light) for 24 hours while maintaining the temperature of 28 ℃, relative humidity of 80% in the growth room. The experiment was repeated twice. The results are shown in Table 2 below.

Germination rate Control Ceracia Marsense N4-5 Primary 43/50 48/50 Secondary 44/50 49/50 Average 43.5 / 50 48.5 / 50 Average germination rate 87% 97%

( Example 3)

Growth promotion experiment of plant by microbe after immersing seed into microbe

Cucumber seedlings germinated from Example 1 and transplanted into a 14 cm wide, 17 cm deep pot containing top soil (Enpico Horticulture Top, Co., Ltd.). Plant growth was given 500 ml of water every three days under 24 hours of white light. Four to five days later, the main leaf emerged, and when the growth reached its highest, the leaf and leaf width were measured. Cucumber seedlings were taken 21 days after sowing to weigh the roots and total weight. The growth conditions were irradiated with artificial light (white light) for 24 hours until the 15th day and maintaining artificial light only for 12 hours from the 16th to the 21st day, maintaining the temperature of 28 ℃, 80% relative humidity in the growth room. The results are shown in Tables 3 to 6 below.

Stem Length (Figure 3) Days after sowing Stem Length (cm) Control Ceracia Marsense N4-5 % Increase Day 4 1.460 2.160 47.94 6th day 5.660 7.500 32.51 8th day 5.775 8.280 43.38 Day 10 6.250 8.500 36.00 Day 12 6.600 8.920 35.15 Day 14 6.900 8.940 29.57 Day 16 7.200 9.340 29.72

Stem Thickness (Figure 4) Days after sowing Stem Thickness (mm) Control Ceracia Marsense N4-5 % Increase Day 4 59.70 59.67 -0.05 6th day 59.73 59.94 0.35 8th day 60.18 60.35 0.28 Day 10 60.48 61.04 0.93 Day 12 60.60 61.06 0.76 Day 14 61.02 61.45 0.70 Day 16 61.41 61.84 0.70

Cotyledon side length (Fig. 5) Days after sowing Cotyledon length (cm) Control Ceracia Marsense N4-5 % Increase Day 4 1.30 1.67 28.46 6th day 1.84 2.26 22.83 8th day 2.41 2.76 14.52 Day 10 3.11 3.42 9.97 Day 12 3.61 3.75 3.88 Day 14 3.65 3.80 4.11 Day 16 3.68 3.80 3.26

Cotyledon leaf width Days after sowing Cotyledon width (cm) Control Ceracia Marsense N4-5 % Increase Day 4 0.68 0.92 35.29 6th day 1.09 1.23 12.84 8th day 1.39 1.61 15.83 Day 10 1.85 2.01 8.65 Day 12 2.06 2.16 4.85 Day 14 2.13 2.22 4.23 Day 16 2.18 2.22 1.83

( Example 4)

Growth promotion experiment by microorganism after immersing root and root zone of seedling with microorganism

Seed cucumber seeds (variety: Cucumis sativus L. Jin Hung Shinpoongcha), take four-day seedlings, and soak the roots and root zones of seedlings in water (control) or in water with a concentration of 10 ⁸ cfu / ml. Horticultural soil, Pungong Co., Ltd.) was transplanted into a 14 cm wide and 17 cm deep pot. Plant growth was given 500 ml of water every three days under 24 hours of white light. Four to five days later, the main leaf emerged, and when the growth reached its highest, the leaf and leaf width were measured. Cucumber seedlings were taken 21 days after sowing to weigh the roots and total weight. The growth conditions were irradiated with artificial light (white light) for 24 hours until the 15th day and maintaining artificial light only for 12 hours from the 16th to the 21st day, maintaining the temperature of 28 ℃, 80% relative humidity in the growth room. This experiment was repeated four times. The management log of cucumber seeds after sowing is shown in Table 7 below.

1 day 2 days Sprout 3 days Cotyledon infestation 4 days Cotyledon Port port Microbial immersion 5 days 6 days 1 main leaf appearance 7 days 8th 9th 2 main leaves 10 days 11th 1 leaf measurement 12 days 13th 3 main leaves 14 days 2 leaf measurement 15th 4 main leaves 16th 5 main leaves 17 days 3 leaf measurement 18 days 19th 20 days 4 leaf measurement Weighing 21st Microbiological testing

2-A. After soaking, seedlings, leaf length, and leaf width of the leaves growing in the pots were measured to determine the amount of leaf growth. The results are shown in Table 8 below.

Pahu Days Iterations Water (Control) Ceracia Marsense N4-5 Second leaf Third leaf Fourth leaf Second leaf Third leaf Fourth leaf Headboard (mm) Leaf length (mm) Leaf width (mm) Leaf length (mm) Leaf width (mm) Leaf length (mm) Leaf width (mm) Headboard (mm) Leaf length (mm) Leaf width (mm) Leaf length (mm) Leaf width (mm) Leaf length (mm) Leaf width (mm) 14 days One 32.00 50.04 58.43 24.00 53.09 58.94 2 24.00 50.08 59.46 25.00 50.09 55.86 3 23.00 47.23 52.22 30.00 56.61 64.83 4 25.00 29.92 30.16 20.00 66.78 57.24 Average 26.00 44.32 50.06 24.75 56.64 59.22 17 days One 36.97 39.49 46.86 47.91 2 36.97 41.40 43.25 45.70 3 41.15 44.62 49.11 52.67 4 18.21 16.00 50.67 59.69 Average 33.32 35.38 47.47 51.49 20 days One 37.74 35.39 46.25 43.84 2 51.50 50.02 58.55 59.12 3 22.97 20.85 52.20 52.75 4 52.85 56.74 61.60 61.02 Average 41.27 40.75 54.65 54.18

2-B. After seedling immersion, the total production of seedlings grown for 17 days in the pot was measured. The results are shown in Table 9 below.

Days after sowing Iterations water Ceracia Marsense N4-5 Headboard (mm) Basis weight (g) Gross weight (g) Headboard (mm) Basis weight (g) Gross weight (g) 21st One 89.19 0.38 7.74 82.84 0.94 8.84 2 79.71 0.61 9.00 83.34 0.47 9.62 3 75.59 0.71 8.07 86.76 0.45 9.45 4 83.53 0.62 6.16 79.18 0.67 10.83 Average 82.01 0.58 7.74 83.03 0.63 9.69

2-C. Table 10 below shows the values obtained by calculating the growth rate for the control (when treated only with water) when seedlings treated with Ceracia marsense N4-5 as a strain according to the present invention.

After sowing Ceracia Marsense N4-5 First leaf Second leaf Third leaf Fourth leaf gun Leaf Leaf width Leaf Leaf width Leaf Leaf width Leaf Leaf width Mojang Basis weight Total amount 11th 10.6% 9.9% 14 days 27.8% 18.3% 17 days 42.5% 45.5% 20 days 32.4% 33.0% 21st 1.2% 8.6% 25.2%

2-D. The number of cells of the new strain Ceracia marsense N4-5 of the present invention that survived around the roots was counted. The results are shown in Table 11 below. The initial microorganisms attached to the rhizosphere were 10 ⁹ cfu, the total microbial count was about 4x10 ⁶ cfu at 17 days after root soaking, and the soil weight of the rhizosphere was 30 g.

Iterations Around the roots Ceracia Marsense N4-5 One soil Per gram of soil 2 x 10 ⁴ Rhizosphere Per gram of soil 2 x 10 ⁵ Rhizoplane Whole root 2 x 10 ⁵ 2 soil Per gram of soil 5 x 10 ² Rootright Per gram of soil 3 x 10 ⁵ Rhizoplane Whole root 9 x 10 ⁵ 3 soil Per gram of soil 1 x 10 ⁴ Rootright Per gram of soil 4 x 10 ⁵ Rhizoplane Whole root 4 x 10 ⁵ 4 soil Per gram of soil 1 x 10 ⁴ Rootright Per gram of soil 4 x 10 ⁵ Rhizoplane Whole root 3 x 10 ⁵ Average soil Per gram of soil 1.2 x 10 ⁴ Total soil (cfu) 1.2 x 10 ⁷ Rootright Per gram of soil 3.2 x 10 ⁵ Whole root (cfu) 9.6 x 10 ⁶ Rhizoplane Per gram of root 6.0 x 10 ⁵ Whole root (cfu) 3.8 x 10 ⁵

From the above results, the first main lobe was inoculated on day 6 after sowing and the growth measured on day 11 was about 10% increase in the new bacterial submerged zone of the present invention compared to the control. However, the second main leaf emerged on the 9th day after sowing, and the measurement of the leaf length and the leaf width on the 14th day showed that the new strain Ceracia marsense N4-5 of the present invention increased the growth of 18 to 28% compared to the control. The third main lobe emerged on the 13th day after sowing, and the growth seen on the 17th day was 42-45% higher than that of the control. The reason why the growth rate of the first leaf is lower than that of other leaves is that the effect of the strain was not 100% or the fertilizer in the soil was promoted, and the growth of the first leaf was promoted. do. As a result of the reduced fertilizer component and the effects of microorganisms, the growth of the second and third leaves was remarkable. From the fourth leaf, growth was reduced, which is thought to be a result of the decrease in the effect of the strain. The total weight of seedlings shown on day 17 after soaking of the strains was increased by 25.2% in the new strain Cerasia marsense N4-5 immersion zone of the present invention. The total number of strains was 10 ⁹ cfu when immersed, and the maximum number of strains was 3x10 ⁶ cfu at 17 days. It is most likely that the strains acted as nutrients to plants by breeding and killing. The presence of the strain in the Rhizoplane as well as in the rhizosphere may add to the possibility of plant soil disease control by habitat occupation. In addition, on the fourth day when the cotyledons were fully grown, the soaking zone was not weakened by fertilizer, while the NPK treatment was 75% (4/4, 3/4, 3/4, 2/4, 3/4, 5 times). Repetition average) showed weakness due to fertilizer. This suggests that the strain is valuable as a fertilizer for soil. That is, when used in the top soil can promote the germination rate more than 10%, increase the growth of cotyledon 20 to 40%, increase the root sticking can increase the growth of seedlings 30 to 40%. And after transplanting, you can get strong seedlings.

( Example 5)

Damping off control rate

The new strain Ceracia marsense N4-5 or N1-8 according to the present invention was applied to test plants with the pathogen Pythium ultimum as a 10% gelatin formulation at about 10 ⁸ cfu per plant seed and the present invention. The degree of suppression of the wearing disease of plants was determined by the new strain. The experiment was repeated twice. The results are shown in Table 12 below. In the table, gelatin is sterile and contains only gelatin preparation, 0 PU is 0 Receiving pathogen spore sac / cc potting mix, 20 PU is 20 Receiving pathogen spore sac / cc potting mix, 50 PU is 50 Receiving pathogen spore sac / cc potting mix Point to. The same letter following the number is not significant (P ≦ 0.05).

Target crop process gelatin N4-5 N1-8 Pathogen Concentration 0 PU 20 PU 50 PU 0 PU 20 PU 50 PU 0 PU 20 PU 50 PU cucumber Plant / pot 5.00A 0.25C 0.38C 5.00A 3.75B 4.38AB 5.00A 4.13AB 5.00A Incidence 0% 95.0% 92.4% 0% 25.0% 12.4% 0% 17.4% 0% Control rate 70.0% 80.0% 77.6% 92.4% Canthalop Plant / pot 4.63 0.50 0.88 4.75 5.00 4.63 4.75 4.88 4.25 Incidence 7.4% 90.0% 82.4% 5.0% 0% 7.4% 5.0% 2.4% 15.0% Control rate 87.6% 72.6% 85.2% 65.0% Mesmelon Plant / pot 3.00 0.50 1.13 2.50 2.50 2.50 4.13 3.50 3.50 Incidence 40.0% 90.0% 77.4% 50.0% 50.0% 50.0% 17.4% 30.0% 30.0% Control rate 50.0% 37.4% 37.4% 24.8% pumpkin Plant / pot 4.63 0.00 0.00 2.50 2.50 2.50 4.50 3.88 4.00 Incidence 7.4% 100.0% 100.0% 50.0% 50.0% 50.0% 10% 22.4% 20.0% Control rate 92.6% 92.6% 80.2% 82.6%

From the above results, it can be seen that both of the new strains of the present invention exhibited 75 to 100% wearing bottle control rate.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments and experimental examples described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the appended claims and their equivalents, rather than the detailed description, are included in the scope of the present invention.

The strains of the present invention are effective for promoting plant growth and controlling incoming diseases.

Claims (2)

Ceracia marsense N4-5 having plant growth promoting and control of stock diseases. Ceracia marsense N1-8 having plant growth promoting and control of stock diseases.