KR101139794B1 - Biological control of plant diseases by mixed Bacillus subtilis M27 and 5M43 - Google Patents

Abstract

The present invention is to isolate and select useful microorganisms, which are domestic resources, to suppress the generation of vegetable microbial disease, to promote the stable production of fresh raw vegetables, and to control various plant pathogens to contribute to safe and high-quality agricultural production technology. A new Bacillus subtilis 5M34 (Accession No. KACC 91488P) is isolated and selected and provides a method for controlling plant pathogens using the same. In addition, the Bacillus subtilis 5M34 strain inhibited the mycelial growth and germination of fungi of Sclerotinia sclerotiorum (Sclerotinia minor ). In combination with Tillis M27 (KACC 91208P) it is significantly inhibited by 86.6%. Therefore, the present invention can function as a biopesticide against phytopathogens, thereby preventing problems caused by residual pesticides, and there is no harm to the environment, and provides a breakthrough biological control method for the preservation of agricultural ecosystems and comprehensive disease management.

Description

Biological control of plant diseases by mixed Bacillus subtilis M27 and 5M43}

It relates to a plant disease control agent using a mixed strain of Bacillus subtilis M27 and 5M34.

The present invention relates to the field of biological control of phytopathogens, and more particularly to a method for controlling fungal nucleus disease occurring during fresh vegetable cultivation using new bacteria.

Recently, interest in biological control of plant diseases is increasing. Biological control is the use of microorganisms or plant extracts to selectively control the target disease without affecting the environment, other plants and killing, instead of using environmentally harmful chemical pesticides in response to increasing consumer demand for safe agricultural products.

Korean Patent Application No. 10-1998-0054040 (KFCC 11070, KFCC 11071) and Korean Patent Application No. 10-1999-0034872 (KCTC 8831P) have a narrow antifungal spectrum problem, and Korean Patent Application No. 10-2002-0050582 (KCTC 10304BP) A patent for antimicrobial activity in a medium using apricot seeds, Korean Patent Application No. 10-2003-0079546 (KCTC 10535BP) is limited to tomato late blight, cucumber anthrax, barley and cucumber powdery mildew. Korean Patent Application No. 10-2001-0029200 (KCTC 0983BP) contains a part of mycobacterial bacteria, but the growth of lactose and Voges-Proskauer reactions and lactose and mannitol were the best in the biochemical properties of this strain. It is different from the strain of the present invention in that respect. The Korean patent application 10-2001-0029204 (KCTC 0985BP) differs from the strain of the present invention in that the lactose reaction and the growth of lactose and mannitol were the most favorable in the use of carbon source in the biochemical properties of this strain. Korean Patent Application No. 10-2006-060114356 (KACC 91208P) has a difference in temperature, pH, lactose reaction, use of carbon source and nitrogen source. Korean Patent Application No. 10-2006-0114933 (KFCC 11356P) has different strain characteristics and application range. Korean Patent Application No. 10-2006-0023130 (KCTC 10911BP) is different because it can grow under anaerobic conditions. It differs in plant growth promoting ability with Korea Patent Application 10-2006-0027002 (KACC 91205P). There is a difference in biochemical properties from Korean Patent Application No. 10-2006-0132268 (KACC 91281P).

On the other hand outside in Bacillus subtilis and GB03 and Bacillus subtilis MBI 600 is gyunhaekbyeong is negative, Bacillus subtilis QST 713 is the present invention and the strain difference in the characteristics has induced resistance (Biocontrol Agents The Manual of, 2004).

Therefore, there is an urgent need for the development of a strain that can act efficiently on other phytopathogens in addition to fungal nucleus disease, and the development of a method of mass culturing the plant.

Bacillus subtilis 5M34 of the present invention has an effect of inhibiting the onset of various pathogens, including mycobacterial germs that occur in the pre-growth period. Therefore, when the Bacillus subtilis 5M34 strain or 5M43 and M27 strain of the present invention is used in combination, it is considered that it is possible to develop a control method capable of efficiently suppressing the disease of the crop without harming the human being.

By separating, selecting, and using useful microorganisms, which are domestic resources, to suppress the occurrence of vegetable microbial diseases, we aim to contribute to the stable production of fresh raw vegetables and contribute to the development of safe and high-quality agricultural product production technology. To develop, the present invention aims to provide a novel Bacillus subtilis 5M34 that can effectively control mycobacterial disease.

Accordingly, the present inventors have completed a combination of Bacillus subtilis 5M34 and Bacillus subtilis 5M34 and M27 after many years of research to complete a control composition for control of vegetable microbial diseases and microorganisms for the same.

The present invention has a plant disease control agent and control method such as fungal nucleus disease using a mixture of Bacillus subtilis 5M34 and Bacillus subtilis 5M34 and M27.

In the present invention, the bacterium was isolated from fermentation, and bacillus subtilis 5M34 strain, which is a bacterium with excellent disease suppression effect, was selected through bioassay. As a result, the bacterium was identified through morphological, biochemical characteristics and DNA profiles comparison. It was identified as Bacillus subtilis 5M34 ( Bacillus subtilis ).

The present invention provides a culture condition of Bacillus subtilis 5M34 strain. For industrial use, a method of culturing the strain in large quantities in a short time is essential. Bacillus subtilis 5M34 strain of the present invention grows well on the TSB medium as a culture medium, 5M34 strain is capable of growing in the range of 10 ~ 52 ℃ and well grown in a wide temperature range of 22 ~ 44 ℃, pH 4.5 ~ 9.5 Growth was possible in the range and the optimum pH was 5.5.

The present invention also provides a phytopathogen control agent prepared using a Bacillus subtilis 5M34 strain, and a mixture of 5M34 and M27 strain, and a method for controlling phytopathogens using the same.

Bacillus subtilis 5M34 strains that can be controlled by the strains that can be controlled Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Sclerotinia minor , Screrotium sepfiborum ( Sclerotium cepivorum , Botrytis cinerea , Rhizoctonia solani and major vegetable pathogens Colletotrichum gloeosporioides , Colletotriccum acutatum , Corynespora cassiicola , Didymella bryoniae , Alternaria solani , Fusarium oxysporum , Phytophthora capsici , etc. This is true. Bacillus subtilis 5M34 strain of the present invention can be controlled by inhibiting the growth of the bacteria.

Gyunhaekbyeong fungus Sclerotinia sclerotiorum was 60.0% in the greenhouse experiments in Bacillus subtilis 5M34 strain processing, Sclerotium minor showed a control efficacy of 53.1%, and a drench treatment intermixing 5M34 and M27 strains gyunhaekbyeong fungus Sclerotinia sclerotiorum was 86.6%, Sclerotium minor showed good control effect of 73.5%.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate an example of the present invention, and the scope of the present invention is not limited to these examples.

The present invention was isolated and cultured new Bacillus subtilis 5M34 strain, deposited in the Korea Agricultural Microorganism Center (KACC) 5M34 strain was given KACC 91488P, the novel Bacillus subtilis 5M34 strain to a variety of plant pathogens Inhibitory effect, especially mycelial growth, mycelium formation, and germ germination of mycobacterial pathogens, and not only treatment with Bacillus subtilis 5M34 strain alone, but also 5M34 and M27 strains showed high control effect in mixed treatment As a replaceable microorganism, it is very safe for agricultural ecosystems and human beings, so it can be effectively used for environmentally friendly control of vegetable microbial disease.

Therefore, the present invention can function as a biopesticide against various kinds of vegetables such as fungal nucleus disease during cultivation can prevent problems caused by crop residues, and has the effect of providing a breakthrough biological control method for the preservation of agricultural ecosystem and comprehensive disease control have.

Example 1 Control Effect on Lettuce Fungal Disease in Greenhouse

Bioassay was inoculated with pathogens by cultivating 4.0 leaves of lettuce in 50-hole tray pot against strains selected by antagonistic investigation, and after 24 hours, isolated microorganisms were incubated in TSB medium for 24 hours and irrigated by 5ml per plant. By examining the condition, a strain (5M34) having excellent bacteriostatic disease suppression effect was finally selected. The Bacillus subtilis 5M34 strain cultured in TSB medium was irrigated 5 ml per lettuce withdrawal, showing 60% control against Sclerotinia sclerotiorum and 53.1% control against Sclerotinia minor . The M27 strain showed control effect at 66.7% for Sclerotinia sclerotiorum and 59.2% for Sclerotinia minor (Table 1). Patent application (Application No. 10-2006-060114356) of strain M27 showed 71.6% of lettuce fungal disease control effect, but in this experiment, the control effect was 66.7%. The control effect may vary depending on the degree of maturation of lettuce and the incidence of no treatment.

[Table 1] Inhibition effect of lettuce fungal disease caused by Bacillus subtilis 5M34 strain (greenhouse)

process

Sclerotinia sclerotiorum

Sclerotinia minor Incidence rate (%) Control price (%) Incidence rate (%) Control price (%) 5M34 20.0 60.0 25.5 53.1 D49 30.0 40.0 38.9 28.5 D75 33.4 31.2 40.0 26.5 DM43 31.1 37.8 42.2 22.4 M27 16.7 66.7 22.2 59.2 No treatment 50.0 - 54.4 -

<Example 2. Mixing effect of Bacillus subtilis M27 strain and other strains>

5M34 including 4 strains M27 mixed with the strain (1: 1) to 10 ⁸ cfu / ㎖ After a concentration by drenching 5㎖ per give lettuce treated with, 5M34 and M27 strains in a mixed treatment than other strains and the mixing treatment, gyunhaekbyeong fungus Sclerotinia The control effect was 86.6% for sclerotiorum and 73.5% for Sclerotinia minor (Table 2). In this experiment, 5M34 and M27 mixed treatment were treated in the same amount once than M27 strain alone to enhance the control effect of 19.9%, which suppressed the occurrence of mycobacterial disease in lettuce cultivation, which would significantly increase the farm income by 19.9%. It has proved to be a very important microorganism.

[Table 2] Inhibitory Effect of Bacillus Subtilis M27 Strains and Other Strains on Lettuce Inhibiting the Occurrence of Lettuce Mycosis (Greenhouse)

process

Sclerotinia sclerotiorum

Sclerotinia minor Incidence rate (%) Control price (%) Incidence rate (%) Control price (%) M27 16.7 66.7 22.2 59.2 5M34 + M27  6.7 86.6 14.4 73.5 D49 + M27 26.7 46.6 35.5 34.7 D75 + M27 30.0 40.0 37.8 30.5 DM43 + M27 26.7 46.6 37.8 30.5 No treatment 50.0 - 54.4 -

Example 3 Isolation, Identification and Culture of Strains

Isolation of the strain was to pulverize the lung surface in order to separate the microorganism from the fermentation obstruction of Suwon, heat-treated at 80 ℃ for 7 minutes, plated in 10% TSA medium and incubated. Through bioassay, the bacterium 5M34 with excellent anti-tumor effect against fungal nucleus was selected.

In the identification method of the strains, the shape, size, shape and spore location were examined, and the physiological investigation was performed with temperature response, pH and various biochemical experiments. Molecular biological identification was performed to identify the nucleotide sequence of 16S rDNA by separating DNA, to identify the phylogenetic location, and to identify it as Bacillus subtilis as a result of DNA-DNA hybridization experiment with the standard strain of Bacillus genus (FIG. 1, FIG. 2, Table 3, Table 4).

Table 3 Results of DNA-DNA hybridization experiment with Bacillus subtilis 5M34 standard strain of Bacillus genus (5M34 is used as a probe)

5M34 Bacillus subtilis
(KACC 10854 ^T ) Bacillus mojavensis
(KACC 12096 ^T ) Bacillus vallismortis
(KACC 12149 ^T ) Bacillus atrophaeus
(KACC 12090 ^T ) Bacillus amyloliquefaciens
(KACC 12067 ^T )
100%
86.0%
18.9%
33.7%
7.7%
2.0%

* Due to DNA-DNA hybridization result, 5M34 is B. subtilis and the DNA showed a high homology (86.%) greater than the bacterial species based on the 70% being identified as B. subtilis.

Table 4 Morphology and biochemical properties of Bacillus subtilis 5M34

Characteristic investigation

5M34 Sporangium bulging Slightly Sphere shape ellipsoidal Sphere position Central Length of cell> 3 μm - Growth at 28 ℃ O Growth at 50 ℃ O Growth at pH 6.0 O Growth with 5% (w / v) NaCl O Anaerobic growth - Degradation of starch O Degradation of casein O Gelatin liquefaction O Utilization of citrate O Nitrate reduction O Oxidase activity O Voges-Proskauer reaction O Acid from L-arabinose O Acid from raffinose O Acid from salicin O Acid from galactose - Acid from lactose - Acid from D xylose w catalase activity O

The growth temperature of the selected Bacillus subtilis 5M347 strain was investigated using a temperature gradient device (Temperature Gradient Incubator TN2148, Advantec.). As a result, the Bacillus subtilis 5M34 strain was grown in the range of 10-52 ° C, and its width was wide. It grew well in the wide temperature range of 22 ~ 44 ℃, and was able to grow in the range of pH 4.5 ~ 9.5 and the optimum pH was 5.5 (Table 5).

Table 5 Growth of Bacillus subtilis 5M34 strain by pH

pH

4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
OD value

1.278
1.330
1.366
1.298
1.163
1.195
1.131
1.156
1.062
0.902
0.863

In order to establish the mass culture conditions of the selected Bacillus subtilis 5M34 strain, the growth of the medium according to the type of medium was carried out using a temperature gradient device (Bio-photo recorder, Advantec Toyo Kaisa, LTD) and the medium type was LB medium (Luria-Bertani). Medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1ℓ, TSB medium (Tryptic soy broth medium: pancreatic digest of casein 17g, enzymatic digest of soybean meal 3g, dextrose 2.5g, sodium chloride 5g, dipotassium phosphate 2.5g Investigation of the growth of bacteria on four culture media, including 1 liter of distilled water, showed that both strains grew well in TSB media (Table 6).

Table 6 Growth of Bacillus subtilis 5M34 by culture medium

Badge

OD value LB 0.861 TSB 1.209 NB 0.329 KB 0.329

<Example 4. Mycelial growth inhibitory effect on phytopathogens>

Antagonist investigation was carried out in the petri dishes in which PDA medium was dispensed in the room and replaced with germs such as fungi and isolated bacteria, and incubated for 10 days at 20-25 ° C depending on the type of pathogen, and then measured the distance to inhibit the fungi. The first selection was made. Bacillus subtilis 5M34 strains include Sclerotinia sclerotiorum , Sclerotium cepivorum , Sclerotinia minor , and Sclerotinium rolfsi (Sclerotium rolfsii), Li Estonian solani (Rhizoctonia solani), Botrytis cine Ria (Botrytis cinerea), heir Solarium oxy Spokane Room (Fusarium oxysporum), Didier in Melaka Brioni (Didymella bryoniae), Alter and Leah solani (Alternaria solani , Corynespora cassiicola , Colletotrichum gloeosporioides , Colletotrichum acutatum , Phytophthora capsici and Poma sp Phoma sp. Appears to inhibit the growth of strains (Table 7).

[Table 7] Inhibitory Effect of Mycelial Growth of Bacillus subtilis 5M34 Strain on Plant Pathogens

Mycelial growth inhibition level

Plant pathogen + Sclerotium rolfsii, Fusarium oxysporum, ++ Sclerotinia sclerotiorum, Sclerotinia minor,
Phytophthora capsici, Botrytis cinerea,
Rhizoctonia solani, Colletotrichum gloeosporioides,
Corynespora cassiicola +++ Sclerotium cepivorum, Alternaria solani, Didymella bryoniae,
Colletotrichum acutatum, Phoma sp .

Note) +, 5 mm or less; ++, 5.1-10 mm; +++, 10.1 mm or more.

<Example 5. Mycobacterium germination inhibitory effect of Bacillus subtilis 5M34 and M27 strains

B. subtilis 5M34, M27, and 5M34 and M27 mixed strains were immersed for 30 min in two strains separated from the culture stock cultured in TSB medium to treat germination of S. sclerotiorum (S. minor ). Treated. The plaque formation was examined in three layers of 10 cells per triangular flask at regular intervals, and irradiated with fluorescent lamps for 12 hours / day in a 15 ° C thermostat. In addition, the direct mycelial germination of mycorrhizal fungi were examined by three repetitions of 10 micropores per 88 mm diameter petri dish on the agar medium treated as above for each microbial bacterium, and then examined for the mycelial germination rate of the microbial germs after 10 days in a 20 ℃ thermostat. It was. In order to investigate the effect of Bacillus subtilis on S. sclerotiorum, S. minor , the germination of S. sclerotiorum was 10%. , S. minors were germinated directly to the mycelium of 20.0%, and follicle formation was not formed in S. sclerotiorum and S. minors , which was more effective than single strains (Table 8).

[Table 8] Inhibition of direct germination and follicular formation of Bacillus subtilis 5M34, M27 strains and mixed strains of 5M34 and M27

Fungi


Treatment concentration
5M34

M27
M27 + 5M34 spawn
Germination rate
(%) Sleeping bag
Formation rate
(%) spawn
Germination rate
(%) Sleeping bag
Formation rate
(%) spawn
Germination rate
(%) Sleeping bag
Formation rate
(%) S.
sclerotiorum 1 × 10 ⁸ ml ^-1 30.0 0   30.0 0 10.0 0 No treatment 100 100 100 100 100 100
S. minor 1 × 10 ⁸ ml ^-1 50.0 0   60.0 3.0 20.0 0 No treatment 100 80.0 100 70.0 100 80

<Example 6. Use of nutrient source of Bacillus subtilis 5M34>

Bacillus subtilis 5M34 strain is the basic data when mass-liquid culture is used to make products by revealing whether carbon source and nitrogen source are nutrient sources, and it shows difference in the characteristics of nutritional availability from other Bacillus subtilis strains. It can also be used as a method of distinguishing from. Bacillus subtilis 5M34 strain of the present invention was investigated for growth of carbon source and nitrogen source. The minimum Bacillus growth medium used in the experiments was sucrose (10.0g), K ₂ HPO ₄ (2.5g), KH ₂ PO ₄ (2.5g), (NH ₄ ) ₂ HPO ₄ (1.0g), MgSO ₄ 7H ₂ O (0.20 g), FeSO ₄ 7H ₂ O (0.01 g), MnSO ₄ 7H ₂ O (0.007 g), distilled water (985 mL). The carbon source and nitrogen source of the medium composition used in the experiment is shown in Table 9, and the growth effect using this composition was measured by absorbance at 620 nm after the growth curve of the microorganisms for 24 hours.

[Table 9] Types of Nutrients

Nutrition

Kinds
Carbon source Ascorbic acid, Cellulose, Dextrin, Fructose, Galactose, Glucose, Glycerol, Lactose, Maltose, Mannitol, Soluble starch, Sorbitol, Sucrose
Nitrogen source (NH _{4) 2} SO _4, Glycine, L-Asparagine, NH ₄ NO ₃ , Peptone, NaNO ₃ , NH ₄ Cl, KNO ₃ , L-Proline, L-Glutamic acid, (NH ₄ ) ₂ HPO ₄ , Yeast extract , Casein, Urea, L-Arginine, L-Alanine

(1) Growth according to the type of carbon source

5M34 strain was investigated by incubating 0.5% of each carbon source except for the carbon source in the Bacillus megaterium growth minimum medium composition. Cellulose was the most effective 5M34 strain, followed by Dextrin, Starch and Fructose.

(2) Growth according to the type of nitrogen source

5M34 strain was examined by injecting 0.5% of each nitrogen source except for the nitrogen source in the Bacillus megaterium growth minimum medium composition. 5M34 strains showed good growth in order of yeast extract, casein and eptone. In the nutrient test for mass cultivation between the two strains, the carbon source Dextrin, Starch, Fructose and nitrogen source Yeast extract, Casein, and Peptone were used.

<Example 7. Antagonistic relationship between Bacillus subtilis 5M34 and M27>

To investigate the antagonistic relationship between Bacillus subtilis 5M34 and M27 strains, 5M34 and M27 strains were incubated in TSB medium at 120 ° C for 2 days at 28 ° C. After culturing the culture medium of M27 in TSB, the TSA medium was cooled to about 45 ° C., mixed with 10 μl of 5M34 medium at regular intervals, and cultured for 3-5 days at 28 ° C. to examine the antagonism of the two strains. . Investigation of antagonism between the two strains proved that there is no antagonistic effect between the two strains is not a problem in the mixing treatment of the two strains (Fig. 3).

Figure 1 shows the phylogenetic location using the 16S rDNA sequence of the novel Bacillus subtilis 5M34 of the present invention.

Figure 2 shows the 16S rDNA nucleotide sequence of the novel Bacillus subtilis 5M34 of the present invention.

Figure 3 shows the co-culture of Bacillus subtilis 5M34 and M27 of the present invention.

<110> Rural Development Administration <120> Biological control of plant diseases by mixed Bacillus subtilis          M27 and 5M43 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1482 <212> DNA <213> Bacillus subtilis 5M34 <400> 1 tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcggacag atgggagctt 60 gctccctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg 120 ggataactcc gggaaaccgg ggctaatacc ggatgcttgt ttgaaccgca tggttcaaac 180 ataaaaggtg gcttcggcta ccacttacag atggacccgc ggcgcattag ctagttggtg 240 aggtaatggc tcaccaaggc aacgatgcgt agccgacctg agagggtgat cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420 gttgttaggg aagaacaagt accgttcgaa tagggcggta ccttgacggt acctaaccag 480 aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 540 ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg 600 gctcaaccgg ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa 660 ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact 720 ctctggtctg taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc 780 ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg 840 ctgcagctaa cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900 ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa 960 gaaccttacc aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg 1020 gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080 cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg 1140 actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200 cctgggctac acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag 1260 ccaatcccac aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct 1320 ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380 caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggta accttttagg 1440 agccagccgc cgaaggtggg acagatgatt ggggtgaagt cg 1482  

Claims (3)

delete A microbial agent for the control of plant pathogens containing the bacteria of the mixed strain of Bacillus subtilis 5M34 (Accession No. KACC 91488P) and Bacillus subtilis M27 (Accession No. KACC 91208P) or its culture solution as an active ingredient. The method of claim 2, The plant pathogens are Sclerotinia sclerotiorum , Sclerotium cepivorum , Sclerotinia minor , Sclerotium rolfsii ), Rhizoctonia solani , Botrytis cinerea , Fusarium oxysporum , Didymella bryoniae , Alternaria solani , Corynespora cassiicola , Colletotrichum gloeosporioides , Colletotrichum acutatum , Phytophthora capsici and Phoma sp. sp.) microbial agent, characterized in that at least one selected from the group consisting of.

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