KR20120084925A - Microorganism agents using trichoderma harzianum ok-1 - Google Patents

Abstract

PURPOSE: A microorganism formulation using Trichoderma harzianum OK-1 strain is provided to promote crop growth and to ensure resistance to various plant pathogen. CONSTITUTION: A microorganism formulation contains Trichoderma harzianum OK-1(KCCM 11097P) and a culture liquid thereof as an active ingredient. The microorganism formulation is used for promoting plant growth or preventing plants.

Description

Microorganism agents using Trichoderma harzianum OK? １}

The present invention relates to a microbial agent, and more particularly, to a plant growth promoter or plant disease control agent using a soil-derived Trichoderma hagianum strain.

Excess chemical fertilizers have been used in agriculture worldwide to provide nutrients that support plant growth. Although chemical fertilizers have provided advantages for modern agriculture, it has been observed that increasing the use of chemical fertilizers reduces the composition and quality of the land. Abuse of chemical fertilizers has caused soil compaction and corrosion, leading to lower production rates and lower fertilizer utilization efficiency.

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

Thus, there is a need for new microbial formulation techniques to establish the compatibility of fertilizer products containing microorganisms.

In addition, many crops are hydroponically grown by genetic engineering techniques during agricultural production. However, when hydroponic farming is carried out, damage caused by late blight and incoming pathogens, which prefer water, has been reported, and most existing pesticides are insoluble in water. It is reported that the control rate is very weak or ineffective.

Therefore, as the interest in environmentally friendly agriculture grows, the interest in controlling methods using biopesticides using microorganisms is increasing. Therefore, development of microbial fungicides using microbial strains or microbial cultures itself is an urgent task.

Thus, the inventors of the present invention, while developing a microbial preparation using soil microorganisms, Trichoderma hagianum strain isolated from domestic soil (Accession Number: KCCM 11097P) acts on the roots of crops to block harmful microorganisms and suitable for growth conditions It improves the environment, promotes growth by increasing the root adhesion of crops, shows excellent water efficiency, and shows resistance to some plant pathogens. The present invention was completed by confirming that it can be usefully used as a microbial preparation for plant growth promotion or plant disease control.

It is an object of the present invention to provide a microbial agent for promoting plant growth or controlling plant diseases using a Trichoderma harzianum strain isolated from soil.

Still another object of the present invention is to provide a method of promoting plant growth or controlling plant diseases using the microbial agent.

In order to achieve the above object, the present invention provides a microorganism agent comprising Trichoderma hazianum OK-1 strain deposited with Accession No. KCCM 11097P or a culture thereof as an active ingredient.

In addition, the present invention provides a method for promoting plant growth, comprising treating the strain, its culture solution or the microbial agent to soil, a plant or a seed of a plant.

In addition, the present invention provides a method for controlling plant diseases comprising the step of treating the strain, its culture or the microbial agent to soil, plants or seeds of plants.

Trichoderma Hajianum OK-1 strain deposited with accession number KCCM 11097P according to the present invention promotes growth by increasing the root adhesion of crops, exhibits excellent water efficiency, and exhibits resistance to various plant pathogens, The strain or its culture may be usefully used as a microbial agent for promoting plant growth or controlling plant diseases.

1 is a diagram showing the results of observing the thick film spores and conidia spores under a microscope before and after homogenization of the Trichoderma hagiannum OK-1 strain according to the present invention.
Figure 2 is a view showing the results of microscopic observation of the thick film spores and conidia spores isolated from the Trichoderma hagihanum OK-1 strain according to the present invention.
Figure 3 is a diagram showing the results of growth promotion experiments for turf of trichoderma hagihanum OK-1 strain according to the present invention.
Figure 4 is a graph showing the results of growth promotion experiments for tomatoes of the Trichoderma hagihanum OK-1 strain according to the present invention.
Figure 5 is a diagram showing the results of experiments to increase the effect on the potato of the Trichoderma hagihanum OK-1 strain according to the present invention.
Figure 6 is a diagram showing the results of experiments on the antimicrobial activity of the gray fungus disease and wearing diseases of the Trichoderma hagihanum OK-1 strain according to the present invention.
Figure 7 is a diagram showing the results of experiments on the antimicrobial activity of anthracnose and wilted disease of the Trichoderma hagihanum OK-1 strain according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a microbial preparation comprising Trichoderma harzianum OK-1 strain deposited with Accession No. KCCM 11097P or a culture thereof as an active ingredient.

The present invention also provides a method for promoting plant growth, comprising the step of treating the soil, the plant or the seed of the plant according to the present invention with a strain, a culture solution thereof, or a microbial agent comprising the same.

In addition, the present invention provides a plant disease control method comprising the step of treating the strain according to the invention, its culture solution or microbial preparation comprising the same to the soil, plants or seeds of plants.

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

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

The plant disease is preferably selected from the group consisting of gray mold disease, wearing disease, anthrax and wilted disease, but is not limited thereto, and includes all plant pathogens.

In an embodiment of the present invention, the Trichoderma hagianum OK-1 strain isolated from the soil (Accession Number: KCCM 11097P) acts on the root of the crop to block harmful microorganisms and improve the environment suitable for growth conditions, the root of the crop By increasing the sticking power to promote growth, showing excellent water efficiency, and showed resistance to some plant pathogens, by separating and cultivating the thick film spores from the strain and confirmed that the mass culture is possible, the strain or its It was found that the culture solution can be usefully used as a microbial agent for plant growth promotion or plant disease control.

The microbial agent according to the present invention can be used not only Trichoderma hagianum OK-1 strain or its culture solution itself, but additionally include an agriculturally acceptable carrier.

The strain according to the present invention, its culture solution, or microbial preparations can be used in the solid state by spraying the soil around the crop, irrigation to plants in liquid state, immersed or sprayed into the seed of the crop, or coated on the seed, but is not limited thereto. .

Strains, cultures or microbial preparations thereof according to the invention can be poured into the soil around the plant (irrigation treatment) or the seeds can be immersed in the culture solution and formulation (immersion treatment) and sprayed (foliar treatment) in the art. The well-known technique can be sprayed to flow through the plant, but is not limited thereto.

The microbial agent according to the present invention is preferably used by diluting the strain according to the present invention or its culture solution by about 10 to 1000 times, and more preferably by diluting about 100 to 500 times.

The microbial agent according to the present invention is preferably sprayed 3 to 10 times at intervals of 3 to 10 days during plant cultivation, and more preferably sprayed 5 times at 7 day intervals, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Isolation of strain

20 g of soil samples collected from a nearby farm in Cheonan, Chungnam, were added to a Erlenmeyer flask containing 100 ml of sterile distilled water and stirred for 30 minutes to prepare a sample solution. After diluting the sample solution to 10 ⁶ ~ 10 ⁷ , take 200 ㎕ of each dilution and spread it on the mold selection medium (Potato Dextrose Agar) and incubate for 5 days at 25 ° C. Single colonies were isolated by subculture in Agar).

Identification of the strain

The isolated strain was inoculated in a basal medium of pH7.0 and incubated at 25 ° C. for 72 hours. Preservation of the strain was stored frozen to include glycerol at a final concentration of 20%. In order to identify the strain by analyzing the ITS rDNA nucleotide sequence of the cultured strain, after extracting DNA from the strain, in order to amplify fragments required for ITS rDNA sequencing, genomic DNA and ITS 1 (5'-) of the strain. PCR reaction was performed using TCCGTAGGTGAACCTGCGG-3 ') (SEQ ID NO: 2) and ITS 4 (5'-TCCTCCGCTTATTGATATGC-3') (SEQ ID NO: 3). PCR reaction was carried out for 1 minute at 95 ℃ for 2 minutes using PCR Thermal cycler TP600 (Takara Co. Japan), 30 seconds at 94 ℃, 30 seconds at 56 ℃, 1 minute at 72 ℃ after a total of 35 cycles 1 cycle was performed for 5 minutes at 72 degreeC. PCR products were purified using QIAquick PCR purification Kit (Qiagen), and then sequenced by Applied Bosystems model 310 automatic DNA sequencer. The nucleotide sequence (SEQ ID NO: 1) of the ITS rDNA thus obtained was analyzed for homology using the BLAST program of NCBI, and the CLUSTAL X program (Thompson et al., 1994) and the PHYLIP program (Felsenstein, 1993) were used. To confirm the systematic location.

As a result, the ITS rDNA nucleotide sequence of the strain was compared with the microorganisms registered in the NCBI GenBank, showing 100% homology with Trichoderma harzianum, confirming the highest similarity, isolated strain Was named Trichoderma Hajianum OK-1.

Therefore, the present inventors deposited the isolates identified and identified in Trichoderma , Korea August 30, 2010 Korea Microorganism Conservation Center deposited as harzianum OK-1 (KCCM 11097P).

Isolation of Conidia and Thick Spores of Strains

Thickral spores and conidia are adhered to the Trichoderma hagianum cells cultured in a liquid incubator, and the cells, thick-film spores and conidia are firstly separated at a low pressure of about 200 bar using a Homogenizer. Using a centrifugal separator at high speed at 8000 rpm to recover the thick film spores and conidia (Fig. 1 and 2).

Measurement of plant growth promoting effect of strains

In order to determine the effect of Trichoderma hagianum OK-1 strain on the growth of grass, cabbage, lettuce and tomato, the strains were treated in pots and fields containing them, and then grown. In the case of grass and tomato, the experiment was conducted using a pot, and the experiment was carried out using horticulture soil (Toby Tech, Woori Soil).

Using the culture medium incubated with the strain in a liquid culture (Fermentor) containing a food source to make the product to a thick film spore concentration of 3.0 X 10 ⁵ cfu / ml or more and diluted 500 times, dipping treatment, irrigation treatment, immersion + Irrigation was performed and compared with no treatment.

In case of turf, put soil on tray (width x length x height: 13 x 20 x 8 cm), and after planting grass, dilute dilute solution 500 times 1L at 1L per 1m ^{2 and} then grow for about 3 months (cm) was measured.

In the case of tomatoes, the dipping process is carried out by diluting the soil seedlings in plastic pots (diameter x height: 30 x 25 cm) and diluting 500 times the tomato seedlings sown in the seedling tray grown for one month. After dipping into 1 L of dilution for 2 to 3 minutes, irrigation was once irrigated with 1 L of dilution per 1 m ² after seeding. Immersion + irrigation treatment was performed once in the above two methods in turn, and the growth length (cm) was measured about one week at intervals of one week after the establishment.

In the case of Chinese cabbage, it was a field experiment conducted by the Plant Protection Research Institute located in Gimpo-si, Gyeonggi-do.After dilution of 500 times diluted cabbage seedlings with 1 L per 1 m ² , after 15 days of irrigation, Live weight (g) was measured.

In the case of lettuce, it is a field experiment conducted by Chungnam National University. Using diluent diluted 500 times of lettuce seedlings, immerse once in 1 L of dilution per 100 weeks for 3 minutes, and after seeding After 1 month of irrigation with 1 liter of diluent per 1 m ^{2, the} length of leaves (cm), leaf width (cm), number of leaves (dog), chlorophyll content (SPAD) and ground weight (g) were measured.

As a result, as shown in Figures 3 and 4, when treated with Trichoderma hagianum OK-1 strain grass and tomato significantly promoted growth 1.3 ~ 1.5 times compared to the untreated control. In addition, as shown in Table 1 and Table 2, the treatment of Trichoderma hagiaumum OK-1 strain increased the fresh weight of Chinese cabbage by 1.2 times compared to the untreated control, 1.1 times (appropriate amount for lettuce) ), 1.3 times (volume) increased growth, the number of leaves increased 1.3 times (titration), 1.5 times (fold), and ground load increased 1.6 times (right), 1.9 times (fold) as well as chlorophyll. The content was also increased by 1.3 times (quantity, doubling) (Figs. 3 and 4, and Tables 1 to 3).

This shows that the promoting effect is good in the batch treatment.

Live weight comparison of Chinese cabbage after strain treatment Treatment method Live weight (g) Soil surface irrigation immediately after planting 52.1a ^{1 )} No treatment 42.8b

Note 1) There is no significant difference between DMRT results at the 5% significance level.

Comparison of Growth Promotion Effects of Lettuce after Strain Treatment Treatment method Throughput ¹⁾ Leaf
(Cm) Leaf width
(Cm) ground game
(dog) Chlorophyll content
(SPAD) Ground load
(g) Soil surface irrigation after dipping 500 times 15.2b ²⁾ 7.7b 10.9a 19.0b 11.3b 250x 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 proper volume, 250 times is double volume.

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

Crop Growth Effect of Strains

<5-1> Validation effect on potato

In order to examine the effect of Trichoderma hagianum OK-1 strain on the increase of potato, the thick film spore concentration was 3.0 X 10 ⁵ cfu using a culture medium in which the strain was cultured with a liquid incubator containing a food source. After making the product to more than / ml diluted 3,000 times, the immersion treatment was carried out compared to the untreated. After immersion in 100 L dilution per 100 kg seed potato in Gangwon-do region for 10 to 20 seconds, dried immediately and settled after 4 to 5 days to measure the yield after cultivation for about 3 months.

As a result, as shown in Figure 5, when treated with the Trichoderma Hajianum OK-1 strain it was confirmed that the yield of the potato is increased by about 130% compared to the untreated control.

<5-2> Verification of the effect of increase in water works

In order to investigate the effect of Trichoderma hagianum OK-1 strain on the increase of rice, the thick film spore concentration was 3.0 X 10 ⁵ cfu using a culture medium in which the strain was incubated with a liquid incubator containing a food source. The product was made to be more than / ml, diluted 500 times, and subjected to topsoil treatment (thick film spores) and immersion treatment (conidia spores and thick film spores) to compare with the untreated. In Chungnam area, 1 liter of dilution per 1 m ² of seedling box was passed about 4 months after surface spraying once in soil, and then the yield per kg (a) of cultivated area was measured.

As a result, as shown in Table 3, when treated with the Trichoderma Hajianum OK-1 strain it was confirmed that the harvest of rice significantly increased by about 5 to 11% compared to the untreated control.

Treatment method Yield (kg / 10a) Quantity index Thick Spore (Topsoil Treatment) 545.7 107 Conidia (Immersion) 534.3 105 Thick Spore (Immersion) 565.0 111 Practice 508.1

<5-3> Effect of Increase in Ginseng

In order to investigate the effect of Trichoderma hagianum OK-1 strain on the growth of ginseng, the culture of the seedling was cultured in a liquid culture (Fermentor) containing the food source after the complete opening of seedlings in Chungnam National University and Sedong After making the product so that the thick film spore concentration was higher than 3.0 X 10 ⁵ cfu / ml, Ginseng (two-year-old root) was cultivated by spraying 500 times dilution 1 L five times at intervals of 7 days per 1 m ² , and after four months The number of harvested individuals per cage (7 rows 10 rows 70 copies transplanted 20 cm planting) was measured.

As a result, as shown in Table 4, when treated with Trichoderma Hajianum OK-1 strain it was confirmed that the yield of ginseng significantly increased by 3 ~ 7.6% compared to the untreated control.

Comparison of Growth Promotion Effects of Ginseng after Strain Treatment Test area division Quantity (g / kan) Quantity index Chungnam National University Treatment 345.1a 107.6 Non-treatment 320.8b - Fibrillation Treatment 359.1a 103.0 Non-treatment 348.7b -

Determination of Antimicrobial Effects of Strains

To investigate the antimicrobial activity of Trichoderma hagiaum OK-1 strains, Etricoderma Hagianum OK-1 strains and phytopathogenic bacteria (Pure fungus, wearing disease, anthrax and Wilt) was replaced and cultured in an incubator at 24 ° C. for 5 days.

As a result, as shown in Table 5, Figure 6 and 7, it was confirmed that the Trichoderma Hajianum OK-1 strain has an excellent control effect on gray mold, wear, anthrax and wilt disease.

Antagonistic effect test against phytopathogenic bacteria Plant pathogen % Inhibition Botrytis cinerea 73.6 Rhizoctonia solani 82 Collectotrichum acutatum 81.5 Fusarium oxysporum 73.33

Korea Microorganism Conservation Center (overseas) KCCM11097P 20100830

<110> K-INSECT CO., LTD. <120> Microorganism agents using Trichoderma harzianum OK-1 <130> DP110125KR <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 555 <212> DNA <213> Trichoderma harzianum <400> 1 gcgggtattc ctacctgatc cgaggtcaac atttcagaag ttgggtgttt tacggacgtg 60 gacgcgccgc gctcccggtg cgagttgtgc aaactactgc gcaggagagg ctgcggcgag 120 accgccactg tatttcgggg ccgggatccc gtcttagggg ctcccgaggt ccccaacgcc 180 gaccccccgg aggggttcga gggttgaaat gacgctcgga caggcatgcc cgccagaata 240 ctggcgggcg caatgtgcgt tcaaagattc gatgattcac tgaattctgc aattcacatt 300 acttatcgca tttcgctgcg ttcttcatcg atgccagaac caagagatcc gttgttgaaa 360 gttttgattc attttgaatt tttgctcaga gctgtaagaa ataacgtccg cgaggggact 420 acagaaaaga gtttggttgg tccctccggc gggcgcctgg ttccggggct gcgacgcacc 480 cggggcgtga ccccgccgag gcaacagttt ggtatggttc acattgggtt tgggagttgt 540 aaactcggta atgat 555 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS 1 <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS 4 <400> 3 tcctccgctt attgatatgc 20

Claims (4)

A microbial agent comprising Trichoderma harzianum OK-1 strain deposited with Accession No. KCCM 11097P or a culture thereof as an active ingredient.
The microorganism preparation according to claim 1, further comprising an agriculturally acceptable carrier, for promoting plant growth or controlling plant diseases.
Trichoderma hagianum OK-1 strain deposited with Accession No. KCCM 11097P, its culture or microbial preparation comprising the same is treated to soil, plant or plant seed, irrigated to plant, soaked in seed of crop or Spraying or coating the seed treatment process comprising the step of treating.
Trichoderma hagianum OK-1 strain deposited with Accession No. KCCM 11097P, its culture or microbial preparation comprising the same is treated to soil, plant or plant seed, irrigated to plant, soaked in seed of crop or A method of controlling plant diseases selected from the group consisting of gray mold disease, wearing disease, anthrax and wilting disease, comprising the steps of spraying or coating the seed and treating the seed.

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