KR20120104875A - Microorganism agents using trichoderma atroviride ob-1 - Google Patents

Abstract

PURPOSE: A Trichoderma atroviride strain and a microorganism formulation using the same are provided to ensure resistance to various plant pathogens. CONSTITUTION: A microorganism formulation contains Trichoderma atroviride OB-1(deposit number KCCM 11173P) or culture thereof as an active ingredient. The microorganism formulation also contains agriculturally acceptable carrier and is used for promoting plant growth or preventing plant diseases. The plant diseases are Botrytis cinerea, Rhizoctonia solani, Collectotrichum acutatum, or Fusarium oxysporum. [Reference numerals] (AA) Growth length(cm); (BB) Before irradiation; (CC) First irradiation; (DD) Second irradiation; (EE) Third irradiation; (FF) Fourth irradiation; (GG) Fifth irradiation; (HH) Sixth irradiation; (II) Seventh irradiation; (JJ) Eighth irradiation; (KK) Ninth irradiation; (LL) Tenth irradiation; (MM) Dipping treatment; (NN) Surface soil treatment; (OO) Dipping treatment + surface soil treatment; (PP) Non-treated group

Description

Microorganism agents using Trichoderma atroviride OB? １}

The present invention relates to a novel microorganism and a microbial preparation using the same, and more particularly, to a novel Trichoderma atroviride strain derived from soil, and a plant growth promoter or plant disease control agent using the microorganism.

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 used, many damages caused by late blight and incoming pathogens, which prefer water, have been reported. 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 agent using soil microorganisms, Trichoderma atroviride strain (Accession Number: KCCM 11173P) isolated from domestic soil acts on the roots of crops to block harmful microorganisms and growth conditions Improved to suitable environment, promoted growth by increasing the root adhesion of crops, and showed excellent water efficiency, and showed resistance to some plant pathogens. By confirming this possibility, 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 novel Trichoderma atroviride strain isolated from soil.

Still another object of the present invention is to provide a microbial agent for promoting plant growth or controlling plant diseases using the novel Tricorderma atroviride strain.

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 Trichoderma atroviride OB-1 strain deposited with accession number KCCM 11173P.

The present invention also provides a microbial agent comprising the strain or its culture 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.

Tricorderma atroviride OB-1 strain deposited with Accession No. KCCM 11173P according to the present invention promotes growth by increasing the root adhesion of crops, exhibits excellent yielding efficiency, and exhibits resistance to various plant pathogens. As such, the strain or its culture may be usefully used as a microbial agent for promoting plant growth or controlling plant diseases.

1 is a view showing the results of observing the thick film spores and conidia spores under a microscope before and after homogenization of the Trichoderma atroviride OB-1 strain according to the present invention.
Figure 2 is a diagram showing the results of microscopic observation of the thick film spores and conidia from the Trichoderma atroviride OB-1 strain according to the invention.
Figure 3 is a diagram showing the results of the growth promotion experiments on the grass of the Trichoderma atroviride OB-1 strain according to the present invention.
Figure 4 is a graph showing the results of the growth promotion experiments for tomatoes of the Trichoderma atroviride OB-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 atroviride OB-1 strain according to the present invention.
Figure 6 is a diagram showing the results of experiments on the antimicrobial activity of the gray mold and wear diseases of Trichoderma atropiriide OB-1 strain according to the present invention.
7 is a diagram showing the results of experiments on the antimicrobial activity of anthracnose and wilted ill disease of the Trichoderma atheroviride OB-1 strain according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a Trichoderma atroviride OB-1 strain deposited with accession number KCCM 11173P.

The strain according to the present invention is preferably derived from soil, but is not limited thereto.

The strain according to the present invention acts on the roots of crops to block harmful microorganisms, improve the environment suitable for growth conditions, increase the root adhesion of the crops to promote growth, characterized in that it has an activity to show an excellent increase efficiency In this case, the crop is preferably grass, cabbage, lettuce and tomato, but is not limited thereto.

The strain according to the invention is characterized in that it has an antimicrobial activity against plant pathogens, wherein the plant pathogens are preferably any one selected from the group consisting of ash fungus, wearing disease, anthrax and wither disease. .

In an embodiment of the present invention, after separating the strain from soil samples collected from a farm near Cheonan, Chungnam, extract DNA from the strain, and amplify the base sequence of the elongation factor (EF) region of the strain using PCR. The homology was analyzed using the BLAST program of NCBI, and the systematic location was confirmed using the CLUSTAL X program and the PHYLIP program. As a result, the nucleotide sequence of the strain showed 99% homology with Trichoderma atroviride compared to the microorganisms registered in NCBI GenBank, showing the highest similarity.

Therefore, it was confirmed that the strain of the present invention is a novel strain, named Trichoderma atropiriide OB-1, and deposited on February 11, 2011 as the deposit name Trichoderma atroviride OB-1 to the Korea Microbiological Conservation Center ( KCCM 11173P).

In another aspect, the present invention provides a microorganism preparation comprising Trichoderma atroviride OB-1 strain deposited with Accession No. KCCM 11173P 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 atropirirate OB-1 strain (Accession Number: KCCM 11173P) isolated from the soil acts on the root of the crop to block harmful microorganisms and improve the environment suitable for growth conditions, crops Increasing the root lubricating power of the to promote growth, exhibited 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, Or it can be seen that the culture medium thereof can be usefully used as a microbial preparation for plant growth promotion or plant disease control.

The microbial agent according to the present invention may not only use Trichoderma atroviride OB-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 elongation factor (EF) region nucleotide sequence of the cultured strain, after extracting DNA from the strain, the strain to amplify the fragment required for EF region sequencing PCR reactions were performed using genomic DNA of EF 1 (5'-ATGGGTAAGGAAGACAAGAC-3 ') (SEQ ID NO: 2) and EF 2 (5'-GGAGGTACCAGTGATCATGTT-3') (SEQ ID NO: 3). . PCR reactions were performed with 20 ng of genomic DNA compared to 30 μl of reaction mixture containing EF-Taq (SolGent, Korea). PCR was performed as follows. Activate Taq-Polymerase at 95 ° C. for 2 minutes, perform 35 cycles at 95 ° C. for 1 minute, perform 1 minute at 55 ° C. and 72 ° C., and then cycle 1 cycle at 72 ° C. for 10 minutes. ) Was performed. The amplified product was purified using Millipore Corp., Bedford, MA, USA, and the sequencing reaction was performed using the PRISM BigDye Terminator v3.1 Cycle sequencing Kit.

Hi-Di formamide (Applied Biosystems, FosterCity, CA) was added to the DNA samples containing the amplified products, incubated at 95 ° C. for 5 minutes, soaked in an ice bucket for 5 minutes, and cooled. ABI Prism 3730XL DNA sequences were analyzed by DNA analyzer (Applied Biosystems, FosterCity, CA). The nucleotide sequence thus obtained (SEQ ID NO: 1) was analyzed for homology using NCBI's BLAST program, and systematically using the CLUSTAL X program (Thompson et al., 1994) and the PHYLIP program (Felsenstein, 1993). The location was confirmed.

As a result, the nucleotide sequence of the strain showed 99% homology with Trichoderma atroviride compared to the microorganisms registered in NCBI GenBank, confirming that it showed the highest similarity. Trichoderma atropiriide OB-1.

Accordingly, the present inventors deposited the isolates identified and identified as Trichoderma on February 11, 2011 to the Korea Microorganism Conservation Center. Deposited with atroviride OB-1 (KCCM 11173P).

Isolation of Conidia and Thick Spores of Strains

Thick-cell spores and conidia are adhered to Trichoderma atheroviride cells cultured in a liquid incubator, and the cells, thick-film spores and conidia are first-order at low pressure of about 200 bar using a Homogenizer Separation, and the thick membrane spores and conidia spores were recovered by rotating at a high speed at 8000 rpm using a centrifugal separator (FIGS. 1 and 2).

Measurement of plant growth promoting effect of strains

In order to determine the effect of Trichoderma atheroviride OB-1 strain on the growth of grass, cabbage, lettuce and tomato, the strains were treated in the pot and the field 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 the Trichoderma atheroviride OB-1 strain grass and tomato significantly promoted the growth 1.3 ~ 1.5 times compared to the untreated control. In addition, as shown in Table 1 and Table 2, when treated with Trichoderma atroviride OB-1 strains, the fresh weight of Chinese cabbage was increased by 1.2 times compared to the untreated control, and in the case of lettuce 1.1 times (Quantity), 1.3 times (double volume) promoted growth, leaf number increased 1.3 times (right volume), 1.5 times (double volume), and ground load increased 1.6 times (right volume), 1.9 times (double volume) In addition, the chlorophyll content was also increased by 1.3 times (quantity, doubling) (Fig. 3 and Fig. 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 the Trichoderma atroviride OB-1 strain on potato growth, the thick film spore concentration was 3.0 X using a culture medium in which the strain was cultured with a liquid incubator containing a food source. After making the product to 10 ⁵ cfu / ml or more and diluted 3,000 times, the immersion treatment was carried out compared to the untreated group. 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 atroviride OB-1 strain strain it was confirmed that the yield of the potato is increased by about 130% compared to the untreated control.

<5-2> At monks  Verification of increase effect

In order to examine the effect of Trichoderma atroviride OB-1 strain on the increase of rice, the thick film spore concentration was 3.0 X using a culture medium in which the strain was cultured with a liquid incubator containing a food source. After making the product to 10 ⁵ cfu / ml or more was diluted 500 times, the topsoil treatment (thick film spores), immersion treatment (conidia spores, thick film spores) was performed 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 atroviride OB-1 strain strain it was confirmed that the harvest of rice significantly increased by 5 ~ 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

To investigate the effect of Trichoderma atroviride OB-1 strain on the growth of ginseng, after the seedlings were completely opened in Chungnam National University and Sedong, the strains were cultured with a liquid culture medium containing a food source. Ginseng (two-year-old root) was cultivated by spraying 5 times of 1 L of 500-fold dilutions every 7 days per 1 m ² after making a product with a thick film spore concentration of 3.0 X 10 ⁵ cfu / ml. After cultivation, the number of harvested individuals per cell (70 rows, 10 rows, 70 cm transplanted) was measured.

As a result, as shown in Table 4, when treated with the Trichoderma atroviride OB-1 strain strain 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

In order to examine the antimicrobial activity of Trichoderma atheroviride OB-1 strain, Petri-dish was used to identify Trichoderma atheroviride OB-1 strain and phytopathogens Receiving disease, anthrax and wilted disease) were replaced and cultured in an incubator at 24 ° C. for 5 days.

As a result, as shown in Table 5, Figure 6 and Figure 7, it was confirmed that the Trichoderma atroviride OB-1 strain strain has an excellent control effect on ash fungus, wearing disease, 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) KCCM11173P 20110211

<110> OBIT CO., LTD <120> Microorganism agents using Trichoderma atroviride OB-1 <130> DP110327KR <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 904 <212> DNA <213> Trichoderma atraviride <400> 1 ggcnnnnnnn ttnnnngttt gcatcctcat tcccctcgtc agcggcattc tctttgccgt 60 tggcactgct gtgtatgaca tttcgctgac catcatcgtc taggggttcg tatttctcca 120 tcacacagct ttcgatcgac tccaatactg acttgctaca ccagccacgt cgactccggc 180 aagtctacca ccgtgagtaa ctcccaattc ctcgagccct accgccatcg actgtgtcgg 240 tcggcgcggg gtatcatcat catgaacgca tccagctgac atttgcccaa tagactggtc 300 acttgatcta ccagtgcggt ggtattgaca agcgtaccat cgagaagttc gagaaggtga 360 gctcatttct gcttttccac aatcatgccc gacaattctg tcctcagtct tgtcattttt 420 ttttctcgca gcatcacacc ccgctttacc tgtctacccc tcctttgcac agcaaaattt 480 tctggctgcc ttgcttggtt tttagtgggg tgccagtttt ttctgtttgg ctggaacccc 540 gctatcgcca ctgtccctcc gtcccaacga attgtcgtcg ctcaattgca tcgtcttctg 600 cctccatctc tgtgtggttc atcgtgctaa tcatgcttca atcaatagga agccgccgaa 660 ctcggcaagg gttctttcaa gtatgcgtgg gttcttgaca agctcaaggc cgagcgtgag 720 cgtggtatca ccatcgacat tgccctctgg aagttcgaga ctcccaagta ctatgtcacc 780 gtcattggta tgtttccagt cttcctcatt gacatttcga ggccatcatt ctaacgtgcc 840 actctacaga cgctcctggt caccgtgatt tcatcaagaa catgtcactg cgggttcana 900 naca 904 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EF 1 primer <400> 2 atgggtaagg aagacaagac 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> EF 2 primer <400> 3 ggaggtacca gtgatcatgt t 21

Claims (5)

Trichoderma atroviride OB-1 strain deposited with accession number KCCM 11173P.
A microbial agent comprising a Trichoderma atroviride OB-1 strain deposited with Accession No. KCCM 11173P or a culture thereof as an active ingredient.
The microorganism preparation according to claim 2, further comprising an agriculturally acceptable carrier, for promoting plant growth or controlling plant diseases.
Trichoderma atroviride OB-1 strain deposited with Accession No. KCCM 11173P, its culture or microbial preparation comprising the same is treated with soil, plant or plant seeds, but irrigated to plants and seed of crops. Plant growth promoting method comprising the step of dipping or spraying or coating the seed treatment.
Trichoderma atroviride OB-1 strain deposited with Accession No. KCCM 11173P, its culture or microbial preparation comprising the same is treated with soil, plant or plant seeds, but irrigated to plants and seed of crops. A method of controlling plant diseases selected from the group consisting of gray mold disease, wearing disease, anthrax disease and wilting disease, comprising the step of dipping or spraying or coating the seed and treating it.

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