KR20140083122A - Chlamydospore formation technology and microorganism agents using trichoderma sp. ok-1 - Google Patents

Abstract

The present invention relates to a microbial preparation using a Trichoderma sp. strain and, more specifically, to a technology of forming a chlamydospore using a Trichoderma sp. OK-1 strain deposited as a deposit no. KCCM 11097P and moreover to an microbial preparation having a chlamydospore formed which can use the strain, the technology of forming a chalamydospore, or a culture medium of the same for the microbial preparation promoting growth of plants or controlling plant disease by increasing the rooting ability of the crops to promote the growth, exhibiting a high yield efficiency, and exhibiting a resistance to various plant pathogens.

Description

Trichoderma sp. [0001] The present invention relates to a technology for forming thick-film spores in OK-1 strains and a microorganism preparation using the same. OK-1}

TECHNICAL FIELD The present invention relates to a thick-film spore-forming technique and a microbial agent, and more particularly, to a soil-derived tricorder sp. And to a plant growth promoting agent or a plant disease controlling agent.

Excessive amounts of chemical fertilizers have been used in agriculture around the world to provide nutrients to support plant growth. While chemical fertilizers have provided benefits to modern agriculture, it has been observed that increased use of chemical fertilizers degrades the composition and quality of the land. The abuse of chemical fertilizers caused compaction and corrosion of the soil, lowering the production rate and lowering the utilization efficiency of the fertilizer.

Thus, recent studies have shown the potential for biological fertilizers using microorganisms. For example, using microorganisms in fertilizers can help to replenish and maintain soil fertility by providing good soil microbial activity, can help to inhibit pathogenic soil organisms, increase plant mass Can help promote microbial activity around the roots to improve the health of the plant and can help release essential nutrients such as nitrogen, phosphorus and potassium and improve soil porosity, water retention and aeration And may help to reduce soil consolidation and corrosion.

Therefore, there is a need for new microbial formulation technology to establish compatibility of fertilizer products containing microorganisms.

In recent years, many crops have been cultivated by genetic engineering techniques in agricultural production. However, in hydroponic cultivation, many kinds of pesticides and foodborne pathogens have been reported to suffer from water, and most conventional pesticides do not dissolve in water. It has been reported that the control rate is very weak or not effective at all.

Therefore, as interest in environmentally friendly agriculture grows in recent years, there is a growing interest in a method of using microbial pesticides. Accordingly, development of a microbial microbicide using the microbial strain or microbial culture itself is an urgent task.

Trichoderma lycoris is a microorganism pesticide, which is practically used as a disease control. Trichoderma lignourum is used as a pesticide for the control of tobacco tobacco. In the United Kingdom and France, microbial pesticides are also registered as active ingredients in Trichoderma harzianum as an active ingredient in vegetables and mold rot.

It is also a representative bacterium of the fungus growth promoting plant (PGPR).

Trichoderma sp. Among these products, most are sprouted conidia and thick spore, and most of them use conidia spores all over the world. In the case of conidiospore, it is easy to form, and it plays a role of the plant activity and the plant disease control agent, but it is most likely to die by itself because it shows sensitivity to the environment.

Therefore, it is necessary to establish the technology of forming thick-film spores and establish the product which is more environmentally adaptive than the conidial spore and can more than double the role of conidial spore.

The thick wall spore is a thick spore formed by the cell wall formed at the tip and middle of mycelium, and it has strong durability because it has a lot of nutrients. There is a very small part in the natural world. It can survive even in the extreme environment. It is possible to survive by collecting the nutrients for the purpose of maintaining the species and covering them with two kinds of shells (cell membranes). Soil persistence is about 5 times that of conidia.

Accordingly, the present inventors have developed a microorganism preparation using a soil microorganism. The strain (Accession No .: KCCM 11097P) acts on the roots of crops to block harmful microorganisms and improve the environment suitable for growing conditions. It promotes the growth by increasing the rooting ability of the crops, The present inventors completed the present invention by confirming that they can be used as a microorganism preparation for accelerating plant growth or for controlling plant diseases rather than conidia by establishing a technique of forming a thick membrane spore from the strain.

It is an object of the present invention to provide a thick-film spore formation technique using a Trichoderma sp. Strain isolated from soil.

Another object of the present invention is to provide a microorganism preparation using the strain, and to provide a method for promoting plant growth or a method for controlling a plant disease using the microorganism preparation.

In order to achieve the above object, the present invention provides a method for forming a thick-film spore using a TRICODERMA sp. OK-1 strain deposited with Accession No. KCCM 11097P and a microbial preparation containing the culture medium as an active ingredient Lt; / RTI >

The present invention also provides a plant growth promotion method comprising the step of treating the strain, the culture liquid thereof or the microorganism preparation to a soil, a plant or a seed of a plant.

The present invention also provides a plant disease control method comprising the step of treating the strain, the culture solution thereof or the microorganism preparation to a soil, a plant or a seed of a plant.

The tricorder spp. Deposited with Accession No. KCCM 11097P according to the present invention. OK-1 strain is used to form thick-membrane spores. It has better environmental adaptability and vitality than conidia and promotes growth by increasing the root-activating power of crops. It exhibits excellent growth efficiency, resistance to various plant pathogens , The strain or a culture thereof can be useful as a microbial agent for promoting plant growth or controlling plant diseases.

Brief Description of the Drawings Fig. OK-1 < / RTI >
Fig. 2 is a graph showing the effect of the present invention on Tricoderma sp. OK-1 strain was observed under an electron microscope and a general microscope.
Fig. 3 is a graph showing the activity of the Tricoderma sp. This study shows the results of experiments on the antimicrobial activity of 10 kinds of gray mold fungi, which are typical plant pathogens such as OK-1, common vegetables.
Fig. 4 is a graph showing the effect of the present invention on Tricoderma sp. This study shows the effect of antimicrobial activity on two species other than Sclerotinia homoeocarpa (Dollar spot), which is representative of OK-1 strain, in grass main bottles.
FIG. 5 is a graph showing the effect of the present invention on the activity of Trichoderma sp. OK-1 strain of the present invention.
Fig. 6 is a graph showing the results of the analysis of Tricoderma sp. OK-1 strain on the turfgrass.
FIG. 7 is a graph showing the activity of the Tricorder sp. OK-1 < / RTI >
Fig. OK-1 strain of the present invention.
Fig. 9 is a graph showing the effect of the present invention on Tricoderma sp. This study shows the results of experiments on the increase effect of OK-1 strain on heat.

Hereinafter, the present invention will be described in detail.

The present invention provides a thick-film spore formation technique of Trichoderma sp. OK-1 strain deposited with Accession No. KCCM 11097P and a microbial preparation containing the culture solution as an active ingredient.

The present invention also provides a plant growth promotion method comprising treating a strain, a culture solution thereof, or a microorganism preparation containing the same according to the present invention to a soil, a plant or a seed of a plant.

The present invention also provides a method of controlling plant diseases, comprising treating a strain, a culture solution thereof or a microorganism preparation containing the same according to the present invention to a soil, a plant or a seed of a plant.

The microbial preparation according to the present invention is preferably used for accelerating plant growth or for controlling plant diseases, but the present invention is not limited thereto.

Preferably, the plant is selected from the group consisting of grass, cabbage, lettuce, tomato, potato, rice field and ginseng, and heat rice, but not limited thereto, and includes all crops.

Preferably, the plant disease is selected from the group consisting of 10 species including gray mold fungus, which is a common vegetable plant disease, and a dollar spot of major lawn disease, but it is not limited thereto and includes all plant pathogens.

In the examples of the present invention, Tricoderma sp. The OK-1 strain (Accession No .: KCCM 11097P) acts on the roots of crops to block harmful microorganisms and improve the environment suitable for growing conditions. It promotes growth by increasing the root engrafting power of the crops, , And it was confirmed that the strain was resistant to some plant pathogens. After confirming that it is possible to mass culture by separating and culturing the thick spore from the strain, the strain or the culture thereof is useful as a microorganism preparation for promoting plant growth or for controlling plant diseases Can be used.

The microorganism preparation according to the present invention is a microorganism preparation comprising Tricoderma sp. The OK-1 strain or its culture itself can be used, as well as an agriculturally acceptable carrier can be additionally used.

The microorganism according to the present invention may be used by spraying a microorganism, a culture solution thereof, or a microorganism preparation in a solid state on the soil around the crop, in a liquid state, in a plant, in a seed of a crop, .

The culture according to the present invention, the culture or the microorganism preparation thereof can be poured (treated with roots) or the seeds can be immersed in the culture solution and the preparation (immersion treatment) in the vicinity of the plant and sprayed (foliar treatment) But are not limited to, spraying to flow through the plant with well known techniques.

The microorganism preparation according to the present invention is preferably used by diluting the strain according to the present invention or the culture solution thereof by about 10 to 1000 times, more preferably about 100 to 500 times by dilution.

The microorganism preparation according to the present invention is preferably sprayed 3 to 10 times at intervals of 3 to 10 days in plant cultivation, more preferably 5 times at intervals of 7 days, but is not limited thereto.

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 contents of the present invention are not limited to the following examples.

Isolation of strain

20 g of soil samples collected from farmhouses near Cheonan City, Chungnam Province were added to the Erlenmeyer flask containing 100 ml of sterilized distilled water and stirred for 30 minutes to prepare a sample solution. After diluting the sample solution to 10 ⁶ to 10 ⁷ , 200 μl of each diluted solution is plated on a Potato Dextrose Agar and incubated at 25 ° C for 5 days. When a dominant fungal colony appears, a potato dextrose Agar) to separate single colonies (Fig. 1).

Identification of the strain

The isolated strain was inoculated in a basic medium of pH 7.0 and cultured at 25 ° C for 72 hours. The stocks were stored frozen in a final concentration of 20% containing glycerol. In order to identify the strain by analyzing the ITS rDNA nucleotide sequence of the cultured strain, DNA was extracted from the strain, and the genomic DNA of the strain and ITS 1 (5'- The PCR reaction was performed using TCCGTAGGTGAACCTGCGG-3 '(SEQ ID NO: 2) and ITS 4 (5'-TCCTCCGCTTATTGATATGC-3') (SEQ ID NO: 3). The PCR reaction was performed for 1 cycle at 95 ° C for 2 minutes using PCR Thermal cycler TP600 (Takara Co., Japan), followed by 35 cycles of 94 ° C for 30 seconds, 56 ° C for 30 seconds and 72 ° C for 1 minute , And 72 ° C for 5 minutes. The PCR product was purified using QIAquick PCR Purification Kit (Qiagen) and sequenced with Applied Bosystems model 310 automatic DNA sequencer. The nucleotide sequence of the thus obtained ITS rDNA (SEQ ID NO: 1) was analyzed using the BLAST program of NCBI and homology was analyzed using the CLUSTAL X program (Thompson et al., 1994) and the PHYLIP program (Felsenstein, 1993) And the systematic location was confirmed.

As a result, the nucleotide sequence of the ITS rDNA of the strain was compared with that of NCBI GenBank. (Trichoderma sp.) And showed the highest degree of similarity. Thus, the isolated strain was identified as Trichoderma sp. OK-1 ".

Therefore, the inventors of the present invention found that the strain isolated and identified above was deposited on August 30, 2010, at the Korean Society for Microbiological Research, Trichoderma sp . OK-1 (KCCM 11097P) (Table 1).

NCBI analysis result Accession Description Max score Total score Overy coverage E-value Max ident AB472078.1 Trichoderma sp. 1026 1026 100% 0.0 100%

Formation of thick membrane spore of strain

Trichoderma sp. Remove some of the cells and incubate for 7 days in primary seeded culture (PDB medium) in Erlenmeyer flask. Secondary mycelial cultivation in Fermentor is carried out for 5 days by using Triton derma sp. Cultured on the appropriate medium (Table 2). Glucose content is measured after 5 days of culture, and when the concentration reaches "0" mg / ml, the medium for forming thick membrane spores (Table 2) is added and thick membrane spores are formed for about 10 days. When the thick-membrane spore concentration reaches about 2 × 10 ⁸ cfu / ml, the culture is stopped and a microorganism preparation is prepared (Table 2, FIG. 2).

Optimal medium composition (secondary mycelium culture medium, thick membrane spore formation medium) division Medium composition Content (g / L) Secondary hypha culture medium Glucose 100 Malt 100 Yeast 10 Medium for forming thick membrane spores Glucose 50 Potassium nitrate 10 KH2PO4 10 Calcium chloride 5

Comparative measurement of thick-spore and conidia

Trichoderma sp. To investigate the survival rate under extreme environmental conditions, the viable temperature and germination rate, agar culture conditions (extreme environmental conditions ) Were measured.

As a result, as shown in Table 3, the survival rate of the thick-membrane spore was more than twice as high as that of the conidiospore even under extreme environmental conditions. In particular, it is possible to survive at 20 to 70 ° C at the viable temperature, so that the survival rate in the soil is high.

Comparative experiment of thick spore and conidia Survival temperature (℃) Mycelial growth rate
(mm / 24h) Germination rate (%)
(Measured on agar medium) Conidia 5 to 30 5 40 Thick spore -20 ~ 70 20.5 90

Strain On plant pathogens  Antibacterial effect measurement

Trichoderma sp. To investigate the antimicrobial activity of the OK-1 strain, a Petri-dish was used to measure the antimicrobial activity of Trichoderma sp. OK-1 and plant pathogens (10 kinds of common vegetables: gray mold fungi and grasses, grass spot: Dollar spot, etc.) were incubated and incubated for 5 days in an incubator at 24 ° C.

As a result, as shown in Table 4, Fig. 3 and Fig. 4, Tricoderma sp. OK-1 strain showed excellent control effect in all plant pathogens, and 90% or more control effect was observed especially in sclerotinia and garlic leaf blight.

Antagonistic effects on plant pathogens Plant Pathogen Inhibition ratio (%) Botrytis cinerea (gray mold) 73.6 Rhizoctonia solani (wearing bottle or constellation) 82.0 Collectotrichum acutatum (anthrax) 81.5 Fusarium oxysporum 73.3 Sclerotinia sclerotiorum 90.0 Fusarium graminearum (wheat red mold) 65.0 Phytophthora cactorum 87.9 Phytophthora cambivora (plague) 68.5 Cylindrocarpon destructans (root rot disease) 78.0 Pleospora herbarum (garlic leaf blight) 95.3 Sclerotinia homoeocarpa (Dollar spot bottle) 65.1 Rhizoctonia solani AG2 -2 (Ⅳ) (Large patchy disease) 79.2 Pythium spp . (Pythium bottle) 81.4

Measuring seed germination promoting effect of strain

Trichoderma sp. In order to investigate the effect of OK-1 strain on the growth of seed, the host and bean sprouts seeds were cultivated in Trico derma sp. The seed germination rate was measured by immersing in the culture medium for 2 hours. In addition, we measured the germination rate after immersion using general water as a control.

As a result, as shown in Table 5 and Fig. 5, in the case of the host and bean sprouts, the germination was completely started from the 3rd day. In the case of the host in the control, germination was not completed in the case of the host, From 5th day it was completely germinated. The germination period showed a difference of about 2 times.

Experiment on Seed Germination Promotion Effect 2days 3days 4days 5days Final Lentil
(host) Control 23% 90% 94% 94% 94% Treat 40% 100% 100% 100% 100% Soybean
(Bean sprouts) Control 32% 90% 95% 100% 100% Treat 50% 100% 100% 100% 100%

Measuring plant growth promotion effect of strain

Trichoderma sp. In order to examine the effect of OK-1 strain on the growth of turf, Chinese cabbage, lettuce, tomato, and radish, the pots and fields containing them were treated with the strains respectively and cultivated. In the case of grass, tomato, and potato, pot experiments were conducted. Experiments were carried out using horticultural soil (Tobietec, Woojoo soil). Field experiments were conducted for cabbage and lettuce.

The above strain was cultivated in a liquid culture medium (Fermentor), and the product was made to have a thick-film spore concentration of 3.0 × 10 ⁵ cfu / ml or more. The product was diluted 500 times and subjected to immersion treatment, .

For grass, the Tray: contains a bed soil to (length x width x height 13 x 20 x 8 cm), a 500-fold dilution by dilution after grass planting in the last three months or after a single infusion treatment with 1L per 1m ² following growth length (cm) was measured.

In the case of tomatoes, a diluted solution of 500-fold diluted tomato seedlings sown on a tray for seedlings grown in plastic pots (diameter x height: 30 x 25 cm) After immersing in 1 L of diluted solution for 2 to 3 minutes, it was treated once with 1 L of diluted solution per 1 m ² after cultivation. The immersion + suspension treatment was performed once in each of the above two methods, and the growth length (cm) was measured for 10 weeks at the interval of one week after the formulation.

In case of Chinese cabbage, it is a field experiment performed by the Plant Protection Research Institute located in Kimpo City, Gyeonggi-do. It is a field experiment of cabbage seedlings after 1 hour of treatment with 1 L per 1 m ² Fresh weight (g) was measured.

In the case of lettuce, it is a Field experiment conducted by Chungnam National University. The experiment was conducted by immersing the lettuce seedlings in a dilution of 500 times with 1 L of diluted solution per 100 minutes for 3 minutes, Leaf length (cm), leaf width (cm), leaf number (number), chlorophyll content (SPAD) and surface area (g) were measured after one month of treatment with 1 L of diluted solution per 1 m ² .

As a result, as shown in Figs. 6 and 7, When OK-1 strain was treated, grass and tomato promoted growth by 1.3-1.5 times compared with untreated control. In addition, as shown in Table 6 and Table 7, in the case of treatment with Trichoderma jadenum OK-1 strain, the live weight of Chinese cabbage was increased by 1.2 times as compared with the untreated control group, and 1.1 times ), 1.3 times more growth, and the number of leaves was increased by 1.3 times (the proper amount) and 1.5 times (by the amount), 1.6 times (the proper amount) and 1.9 times (the number of times) And the content was increased by 1.3 times (proper amount, multiplication). This shows that the disposal process has a facilitating effect. (Figs. 6 and 7, and Tables 6 to 7).

Comparison of fresh weight of Chinese cabbage after strain treatment Processing method Fresh weight (g) Soil surface treatment immediately after formaldehyde treatment 52.1a ^{1 )} No treatment 42.8b

Note 1) DMRT results at the 5% significance level between the same characters are not significant.

Comparison of Growth Promoting Effect of Lettuce after Strain Treatment Processing method Throughput ¹⁾ Leaf
(cm) Leaf width
(cm) ground game
(dog) Chlorophyll content
(SPAD) Above ground
(g) Soil surface treatment after immersion 500 times 15.2b ²⁾ 7.7b 10.9a 19.0b 11.3b 250 times 15.9b 8.6b 12.4b 19.7b 14.0b No treatment 13.5a 6.7a 8.2a 15.0a 7.2a

Note 1) 500 times is the proper amount, 250 times is the volume.

Note 2) There is no difference in DMRT results between the same characters at 5% significance level.

Effect of strain growth on crops

<8-1> Verification of growth effect in potato

Trichoderma sp. To investigate the effect of OK-1 strain on the growth of potato, the strain was cultured with a fermentor in a liquid culture medium (Fermentor) to produce a thick spore concentration of 3.0 × 10 ⁵ cfu / Diluted and subjected to immersion treatment and compared with untreated water. After being immersed in 100 L of 100 g of seed potatoes for 10 ~ 20 seconds in Gangwon-do area, it was dried immediately and after 4 ~ 5 days, it was cultured for about 3 months and yield was measured.

As a result, as shown in Fig. 8, When OK-1 strain was treated, the potato yield was increased by about 130% compared to the untreated control.

<8-2> In the monarchy  Verification of increase effect

Trichoderma sp. To the OK-1 strain examine the effects of the flooding of rice, to be made using the culture broth of the strains in a liquid culture medium (Fermentor) containing the food source is chlamydospore concentration of 3.0 X 10 ⁵ cfu / ml or more products And then diluted to 500 times, and then treated with topsoil (thick spore), immersion (conidia, thick spores) and compared with control. In Chungnam area, 1 L of diluted solution per 1 m ² of seedling box was sprayed onto the soil once, and after 4 months, the yield (kg) per 10 arcs (a) of cultivation area was measured.

As a result, as shown in Table 8, Tricoderma sp. In the case of OK-1 strain, the yield of rice was significantly increased by 5 ~ 11% as compared with the untreated control.

Processing method Yield (kg / 10a) Quantity Index (%) Thick spore (topsoil treatment) 545.7 107 Conidia spores (immersion) 534.3 105 Thick spore (immersion) 565.0 111 Practice Area 508.1

<8-3> Effect of Growing Effect on Ginseng

Trichoderma sp. In order to investigate the effect of OK-1 strain on the growth of ginseng, the culture was incubated in a liquid culture medium (fermentor) containing the feedstock, 3.0 x 10 ⁵ cfu / ml or more, and 1 L of 500-fold diluted solution was sprayed five times at intervals of 7 days per 1 m ^2. After cultivation for 4 months, The number of individuals harvested per plant (70 transplants in 20 cm planting distance) was measured.

As a result, as shown in Table 9, Tricoderma sp. When OK-1 strain was treated, the yield of ginseng was significantly increased by 3 ~ 7.6% compared to the untreated control group.

Comparison of Growth Promotion Effect of Ginseng after Strain Treatment Test area division Quantity (g / square) Quantity Index (%) Chungnam National University Treatment 345.1a 107.6 Non-treatment 320.8b - Femur Treatment 359.1a 103.0 Non-treatment 348.7b -

<8-4> Verification of the effect of increase in heat

Trichoderma sp. In order to investigate the effect of OK-1 strain on the growth of heat, in the case of heat, the seedlings were placed in plastic pots (diameter x height: 30 x 25 cm) Was immersed in 1 L of diluted solution per 100 weeks for 2 to 3 minutes. After the seedling treatment, the suspension was treated once with 1 L of diluted solution per 1 m ² after seedling settling. Body weight was measured after about 2 months after the meal.

As a result, as shown in Table 10, the increase rate was 122% higher than the control.

Comparison of growth promoting effects of heat treatment after strain treatment Average fresh-weight (g / ea) Index (%) Treat 97.3 122 Control 79.7 -

Claims (4)

Thick film spore formation technique using Trichoderma harzianum OK-1 strain deposited with KCCM 11097P.
A microorganism preparation characterized by further comprising an agriculturally acceptable carrier utilizing a strain of Trichoderma harzianum OK-1 deposited under accession number KCCM 11097P, and for promoting plant growth or for controlling plant diseases.
&Lt; tb &gt;&lt; SEP &gt; TCC &lt; SEP &gt; Comprising treating the soil, plant or plant seeds with a strain of OK-1, a culture thereof, or a microorganism preparation containing the same, wherein the soil is planted in a liquid state, immersed in or sprayed with seeds of the crop, Plant growth promotion method.
&Lt; tb &gt;&lt; SEP &gt; TCC &lt; SEP &gt; Comprising treating the soil, plant or plant seeds with a strain of OK-1, a culture thereof, or a microorganism preparation containing the same, wherein the soil is planted in a liquid state, immersed in or sprayed with seeds of the crop, A method for controlling a plant disease selected from the group consisting of 10 species including common plant pathogenic fungi, gray spot fungus, and dollar spot other than grass main plant disease.

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