KR102131456B1 - Microbial agent for promoting plants growth comprising novel mucor circinelloides and method for promoting plants growth using the same - Google Patents

Abstract

The present invention is a new isolated Mucor sircinelloides ( Mucor) circinelloides ) FBTF-14331 strain, a microbial agent for promoting plant growth comprising the strain and a method for promoting plant growth using the same, more specifically, is a novel fungus isolated from roots of grass inhabiting domestic road incisions Mucor circinelloides ( Mucor circinelloides ) FBTF-14331 relates to a microbial agent for promoting growth of plants containing the strain and a method for promoting growth of plants using the same. The microbial agent for promoting plant growth of the present invention and the method for promoting plant growth using the microbial agent are eco-friendly, and can be effectively applied to greening of incision slopes, and thus have an excellent effect in preventing ecosystem destruction and environmental pollution.

Description

MICROBIAL AGENT FOR PROMOTING PLANTS GROWTH COMPRISING NOVEL MUCOR CIRCINELLOIDES AND METHOD FOR PROMOTING PLANTS GROWTH USING THE SAME}

The present invention is a new isolated Mucor sircinelloides ( Mucor) circinelloides ) FBTF-14331 strain, a microbial agent for promoting plant growth comprising the strain and a method for promoting plant growth using the same, more specifically, is a novel fungus isolated from roots of grass inhabiting domestic road incisions Mucor circinelloides ( Mucor circinelloides ) FBTF-14331 relates to a microbial agent for promoting growth of plants containing the strain and a method for promoting growth of plants using the same.

Generally, in various civil engineering works, rocky slopes, such as rocky slopes formed by cutting and filling, or rock incision due to blasting, usually have steep slopes, so the risk of collapse is very high, and various slopes are considered in consideration of the environment with the surrounding landscape. Planting and greening methods are used. However, in the case of such slopes, the ground conditions or the shape of the soil have considerably separated conditions for the vegetation to grow naturally, so that the vegetation is established spontaneously in the early stage, or the trees firmly settle and propagate on the ground and grow into green forest like the surroundings. Since it is difficult to realistically expect to lose, in the case of soils due to cutting or embankment among these slopes, vegetation seeds are sprayed with artificial soil mixed with them, or the rock incision with severe cracks and unstable working conditions In such cases, it is common to perform artificial recording by striking a net on a rock incision surface, anchoring it, preparing it for loss, and then attaching artificial soil mixed with seeds on the net.

On the other hand, in recent years, many researchers have tried to change the microbial phase in the soil in order to advantageously use it for plant growth. Bacterization refers to the inoculation of microbial cultures into crop seeds or growth sites, and these inoculations sometimes result in increased crop yields, but it is still unclear what role the inoculated microorganisms actually play in the root area. Does not. Depending on the type, if there is a type that maintains its density by stably surviving and growing in the root area within the crop growth period, some may not be settled in the root area by being rejected by the original existing root area microorganism. At this time, microorganisms that stably settle and multiply around the roots are called myosphere microbes. Microbial rooting is a very active process, and is a whole process in which microorganisms multiply using seed secretions rich in carbohydrates and amino acids while living on the surface of the seed or in the soil, and continue to move and grow along the growing roots by attaching at the root surface. . In addition, the myosphere microbe may be passively moved to the lower part of the root by irrigation. Pseudomonas microorganisms include Pseudomonas, Azotobacter, and Bacillus. Myopathic microorganisms have a high growth rate, motility, and tend to prefer root secretions. do. Previously, the beneficial effects of plant growth-promoting rhizosphere microorganisms were recognized only for root crops such as radish, potato, and sugar cane, but recently, positive effects have been recognized in various crops such as oats, soybeans, cotton, peanuts, balsam sprouts, rice, and vegetables. .

Accordingly, there is a need to develop an effective method to effectively improve the growth of a herbaceous plant in an incision site having a high risk of collapse by applying a myosphere microorganism having a plant growth promoting effect to an incision site slope, and to achieve harmony with the surrounding progress.

KR 10-0713059 B KR 10-1066283 B

The present invention has been devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to isolate a new microorganism having a plant growth promoting activity existing at the root of an herbaceous plant inhabiting an incision site and It is to identify and use this as a microbial agent for promoting plant growth, and to provide a method for promoting plant growth using the microbial agent.

In order to solve the above problems, the present invention provides a Mucor circinelloides FBTF-14331 designated as accession number KCTC13414BP having a plant growth promoting activity including germination promoting.

Further, the present invention provides a microbial agent for controlling plant growth promoting containing Mu cor when unsealing nelroyi des (Mucor circinelloides) FBTF-14331 strain is designated as deposit number KCTC13414BP.

The strain is preferably in the form of a strain culture.

The plant is preferably selected from the group consisting of ant odor, sticky sprouts, cypresses, vultures, wild plants, cornflowers, wormwood, rapeseed, sage, coriander, tall fescue and perennial glass.

In addition, the present invention provides a method for promoting plant growth, comprising spraying the microbial agent of the present invention onto a plant seed, leaf, stem or root.

The microbial agent for promoting plant growth of the present invention and the method for promoting plant growth using the microbial agent are eco-friendly, and can be effectively applied to greening of incision slopes, and thus have an excellent effect in preventing ecosystem destruction and environmental pollution.

1 shows a grass as a sample collection plant for isolating Mucor circinelloides FBTF-14331, which is a new microorganism of the present invention, accession number KCTC13414BP.
2 shows a pure isolated strain colony of Mucor circinelloides FBTF-14331, a novel microorganism of the present invention, deposited under accession number KCTC13414BP.
Figure 3 is a new microorganism of the present invention, the deposit number KCTC13414BP Mucor Circinelloides ( Mucor circinelloides ) FBTF-14331 is a graph showing the growth promoting effect of dwarf rice.
FIG. 4 is a graph showing the germination promoting effect of non-repairing seeds of a culture of Mucor circinelloides FBTF-14331, which is a new microorganism of the present invention, accession number KCTC13414BP.
5 is a photograph showing the effect of promoting germination of unrepaired seeds of a culture of Mucor circinelloides FBTF-14331, designated as the new microorganism of the present invention, accession number KCTC13414BP.
Figure 6 is a new microorganism of the present invention, the deposit number KCTC13414BP Mucor Circinelloides ( Mucor circinelloides ) FBTF-14331 showing the effect of promoting the germination of seeds of the sticky sprouts of the culture.
7 is a photograph showing the effect of promoting germination of sticky sprouts seeds of Mucor circinelloides FBTF-14331 culture, designated as the new microorganism of the present invention, accession number KCTC13414BP.
Figure 8 is a new microorganism of the present invention, the deposit number KCTC13414BP Mucor Circinelloides ( Mucor circinelloides ) FBTF-14331 showing the germination promoting effect of seeds in the culture of the seed culture.
9 is a photograph showing the effect of promoting germination of toluskew seeds in a culture of Mucor circinelloides FBTF-14331, designated as the new microorganism of the present invention, accession number KCTC13414BP.
FIG. 10 is a graph showing the germination promoting effect of rapeseed seeds of a culture of Mucor circinelloides FBTF-14331, designated as the new microorganism of the present invention, accession number KCTC13414BP.
FIG. 11 is a photograph showing the effect of promoting germination of rapeseed seeds in a culture of Mucor circinelloides FBTF-14331, which is designated as a new microorganism of the present invention, accession number KCTC13414BP.
FIG. 12 shows the optimal growth temperature of Mucor circinelloides FBTF-14331, a novel microorganism of the present invention, deposited under accession number KCTC13414BP.
FIG. 13 shows the optimal growth pH of Mucor circinelloides FBTF-14331, a novel microorganism of the present invention, deposited under accession number KCTC13414BP.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention utilize the technology to develop biological resources such as river wetlands, coastal wetlands, mountain wetlands, and dune wetlands that make up a special bio-environmental ecosystem to separate microorganisms that symbiotic in the incisal plant roots, and By developing a product that facilitates the interaction between plants and microorganisms by researching a stable formulation method that can be stored and stored, the ecosystem is efficiently maintained and managed, and ecosystems such as incisions that are rapidly damaged by global warming and human activities are developed. It was intended to develop a plant growth-promoting microbial agent capable of efficient restoration.

Accordingly, the present invention has a plant growth promoting activity including germination promoting Mucor Circinelloides (Depot No. KCTC13414BP) circinelloides ) FBTF-14331.

The Mucor sirsinelloides of the present invention circinelloides ) FBTF-14331 strains have been deposited on December 5, 2017 with deposit number KCTC13414BP at the Korean Collection for Type Cultures (KCTC).

The new microorganism deposited with the accession number KCTC13414BP of the present invention, Mucor circinelloides FBTF-14331 strain is excellent in plant growth, particularly root growth and germination promoting effect of seeds.

Accordingly, the present invention provides a microbial agent for promoting plant growth, comprising the strain Mucor circinelloides FBTF-14331, designated as deposit number KCTC13414BP.

The strain is preferably in the form of a strain culture. The strain culture solution is preferably prepared by culturing the strain in a potato dextrose broth (PDB) medium under conditions of 20 to 30°C and pH 4 to 8.

The plant is not particularly limited, but is suitable for greening incision slopes, for example, ant odor, sticky sprouts, cypresses, vultures, wild lands, cornflowers, sagebrush, oilseed rape, sage, irises, tolfesque and perennial It is preferably selected from the group consisting of allied glass.

In addition, the present invention provides a method for promoting plant growth, comprising spraying the microbial agent of the present invention onto a plant seed, leaf, stem or root.

The spraying may be applied directly to the plant or around the plant, and for example, spraying or the like may be used.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples merely describe one preferred embodiment of the present invention, and it is clear that the scope of the present invention is not limited by the matters described in the following examples.

[ Example ]

Example 1. Selection of native plants

As a plant that can be applied to restore slopes such as incision in domestic roads, and examining plant species that can inhabit the slopes, weeds, irises, squirrels, antlers, vultures, mongcho, chamsari, mountain, sticky herbs, Geumgye-guk, Sorae 11 native species such as chrysanthemum and turf were selected, and plant species native to Pohang, Gumi, Gyeongsan and Jinju, Gyeongsangbuk-do were collected and used to separate microorganisms present in the root zone (Fig. 1).

Example 2. Pure separation of microorganisms from collected plants

After removing the soil from 11 kinds of plant roots native to the selected domestic slope incision, put it in distilled water containing Tween 80, shake it at 25°C and 180 rpm for 10 minutes, wash it with distilled water, and add 1% perchloric acid to the roots. After shaking for 2 minutes at 25°C and 150 rpm for 20 minutes, the mixture was washed with distilled water. The washed roots were incubated for about 7 days at 25°C by placing the roots on a Hagem Minimal Agar plate (MH medium) containing Streptomycin. After cultivation, the fungus growing after obtaining the part that does not come into contact with other fungi, streking it in HM medium, incubating for about 7 days again at 25°C, and subcultured single colony in PDA medium to 23 species in Pohang corolla, Gyeongbuk, Gyeongbuk 20 species in Pohang mugwort wormwood, 21 species in Ganbukpo, Gyeongbuk Pohang, 24 species in Visu-ri, Pohang, Gyeongsangbuk-do, 25 species in Nang-Ao, Gyeongnam Jinju, 20 species in Jinju Chamsari, 17 species in Jinju, Gyeongnam, 22 species in Gumi, Gyeongbuk, Gyeongbuk A total of 265 microorganisms were isolated by separating 21 species from Gumi chamsari, 24 species from Gumi sticky ginseng, 21 species from Bisuri, Gyeongsan, and 27 species from Gyeongsan grass, Gyeongbuk. The isolated strain was tested while preserving at -70°C (FIG. 2).

Example 3. Microbial identification through sequencing of purely isolated endogenous fungi

For identification of strains isolated from 11 harvested plants, bidirectional analysis was performed with ITS1 (SEQ ID NO: 1) forward and ITS4 (SEQ ID NO: 2) reverse for the cultured strains. The sequence analysis was completed using the Finch TV program and NCBI blast, and the cells that confirmed the sequence were identified using NCBI blast. Among the identified strains, FBTF-14331 isolated from roots of grass obtained from Gyeongsan, Gyeongbuk, was confirmed to be Mucor circinelloides by the sequencing analysis results shown in Table 1 below.

[Table 1] Isolation strain FBTF -14331 nucleotide sequence (SEQ ID NO: 3) analysis results

Example 4. Culture of isolated strains and sample preparation

Isolation strain Mucor sircinelloides circinelloides ) FBTF-14331 was inoculated into a 250 ml Erlenmeyer flask containing 100 ml of Czapek liquid medium and cultured for 10 days at 25°C and 180 rpm. After 30 ml of the culture solution was cultured and frozen at -70°C for 24 hours, freeze-dried for 5 days, and after freeze-drying, dissolved in 1 ml of primary distilled water and used as a sample.

Example 5. Verification of plant growth promotion effect

Isolation strain Mucor sircinelloides circinelloides ) In order to verify the effect of promoting plant growth of FBTF-14331, an effect was tested using waito-c. The seeds of the wadded rice (Waito-c) are washed 2-3 times with distilled water, placed in DW 2,000ml, mixed with 100 μl of Forcedac and 4 μl of Uniconazole, overnight at 30° C., washed until clear water comes out. Used. The seeds of the ovary rice (waito-c) are germinated in a medium of 0.6% pure agar, and when germination is confirmed, they are inoculated into two mediums and cultured for about 5-7 days in a chamber where 30°C and 20°C are controlled for 12 hours. To verify the effect, 10 μl of the strain culture obtained from Example 4 was inoculated into the leaves of the plant for germinated plants and cultured for 5 days to measure the length of the entire plant and the length of the roots to verify the effect, and as a result It is shown in Table 2 and Figure 3 below.

[Table 2] Dwarf rice Growth promotion effect

As shown in Table 2 and Fig. 3, the isolated strain Mucor circinelloides FBTF-14331 improved the root growth by 48.16% and the overall plant growth by 29.07%.

Example 6. Verification of seed germination effect

Isolation strain Mucor sircinelloides circinelloides ) In order to verify the seed germination effect of FBTF-14331, experiments were carried out by securing unrepaired, sticky greens, tall husks and rapeseed seeds. For the experiment, 100 seeds of each were immersed in distilled water for one day, followed by a control (Con) and an experiment ( Mucor). circinelloides FBTF-14331 culture medium (1 ml), spread gauze on 2 petri dishes, spread 100 seeds of distilled water, 1 ml of distilled water daily in the control, and Mucor in the laboratory. while adding circinelloides FBTF-14331 culture 1ml was measured by the number of the week day germinated seeds. As shown in Figures 4 to 11, all four seeds showed excellent results in the number of germination seeds and growth rate.

Example 7. Exploration of growth conditions of selected strains

Isolation strain with proven plant growth promoting effect Mucor circinelloides ( Mucor circinelloides ) FBTF-14331 In order to explore the growth conditions, the pre-cultured strains were cut into 1 cm each of the horizontal and vertical plates, and then sterilized 3 pieces Inoculated in 300 ml of the prepared PDB medium, the cells cultured with shaking at 25°C and 140 rpm for 5 days were used as seed strains, and the cultured strains were stored frozen at -70°C for experiments. In order to measure the optimal temperature for mycelial growth of the isolate strain Mucor circinelloides FBTF-14331, 4 250 ml Erlenmeyer flasks containing 100 ml of PDB medium were inoculated with 3% of the seed, 20 °C, 25 °C, Shaking at a rate of 140 rpm in four temperature sections of 30° C. and 37° C., followed by incubation for 5 days to measure the amount of cells. The amount of cells was filtered by filtering the culture medium on a filter paper, and the weight was measured. As a result, the best growth was confirmed at 30°C. At 37°C, the amount of cell formation was rapidly decreased (Table 3).

[Table 3] Measurement of optimum temperature for growth

In addition, in order to measure the mycelial growth degree according to the pH change at the optimum temperature of 30°C, the pH of the medium was adjusted to pH 4, pH 5, pH 6, pH 7 and pH 8 using 1N NaOH and 1N HCl in 100 ml of PDB medium. After adjusting and inoculating 3% of seed germs and cultivating for 5 days, the best growth was observed at pH 6, and the production rate of the cells decreased as the acidity and alkalinity increased.

[Table 4] Optimal pH measurement for growth

Example 8. Pilot culture and prototype use

The optimal growth conditions were confirmed by verifying the growth conditions of the selected strains, and the pilot culture was performed under the optimal growth conditions using a 5L fermenter. Liquid and powder formulations that can be applied in the field were prepared using the strains in which the pilot culture was completed, and used in the field application.

<110> FARMBIOS Inc. <120> MICROBIAL AGENT FOR PROMOTING PLANTS GROWTH COMPRISING NOVEL MUCOR CIRCINELLOIDES AND METHOD FOR PROMOTING PLANTS GROWTH USING THE SAME <130> YP170048 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS1 forward primer <400> 1 tccgtaggtg aaccttgcgg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS4 reverse primer <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 496 <212> DNA <213> Artificial Sequence <220> <223> FBTF-14331 rDNA seq <400> 3 tgtattatta cttgacgctt gagggatgct ccactgctat aaggataggc ggtggggatg 60 ctaaccgagt cataatcaag cttaggcttg gtatcctatt attatttacc aaaagaattc 120 agaattaata ttgtaacata gacctaaaaa atctataaaa caacttttaa caacggatct 180 cttggttctc gcatcgatga agaacgtagc aaagtgcgat aactaatgtg aattgcatat 240 tcagtgaatc atcgagtctt tgaacgcaac ttgcgctcat tggtattcca atgagcacgc 300 ctgtttcagt atcaaaacaa accctctatc caacattttg ttgaatagga atactgagag 360 tctcttgatc tattctgatc tcgaacctct tgaaatgtac aaaggcctga tcttgtttga 420 atgcctgaac ttttttttaa tataaagaga agctcttgcg gtaaactgtg ctggggcctc 480 ccaaataata cttttt 496

Claims (5)

delete A microbial agent for promoting germination of herbaceous plants for slope recording, including the strain Mucor circinelloides FBTF-14331, designated as deposit number KCTC13414BP. 3. The method of claim 2, wherein the herbaceous plant for slope recording is selected from the group consisting of ant odor, sticky sprouts, cypress, non-eagle, wild-flowered, cornflower, wormwood, rapeseed, celery, coriander, toll fescue, and perennial glass. Microbial preparation characterized in that. delete delete

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