KR20130101309A - Methylobacterium sp. cbmb12 strain having controlling activity against bacterial spot disease of tomato and uses thereof - Google Patents

Abstract

PURPOSE: A method for preventing bacterial spot in tomatoes using Methylobacterium sp. CBMB12 is provided to promote the growth of the crops and to increase production yield by a natural eco-friendly method without toxicity. CONSTITUTION: Methylobacterium sp. CBMB12 (KACC 91707P) prevents bacterial spot in tomatoes. A composition for preventing bacterial spot in tomatoes contains the strain or a culture liquid thereof as an active ingredient. The pathogen causing bacterial spot is Xanthomonas campestris pv. Vesicatoria. A method for preventing bacterial spot in tomatoes comprises the step of spraying effective amount of Methylobacterium sp. CBMB12 to plants or soil.

Description

Methylbacterium sp. With tomato spot control activity. CMC12 strain and its use {Methylobacterium sp. CBMB12 strain having controlling activity against bacterial spot disease of tomato and uses approximately}

The present invention is a methylbacterium sp. The present invention relates to a CBMB12 strain and a use thereof, and more particularly, to methylcobacterium sp. CBMB12 strain, the composition for the control of the bacterium bacterium bacterium comprising the strain or its culture as an active ingredient, the methylobacterium sp. Method for controlling the bacterium bacillus by spraying an effective amount of the CBMB12 strain on plants or soil and the methylobacterium sp. The present invention relates to a method for preparing a composition for controlling antibacterial bacteria of tomato, comprising culturing the CBMB12 strain.

In various environments, plants are exposed to physical, chemical and / or biological stresses, among which, stress and damage caused by pathogens corresponding to biological stresses are very widespread unlike other environmental factors. Plants exposed to these pathogens release ethylene in their initial reactions, and excessive release of ethylene causes symptoms of the disease. Therefore, the activity of ACC deaminase that hydrolyzes ACC (1-aminocyclopropane-1-carboxylate), which is a precursor of ethylene, and the microorganisms producing the same, may maintain stress ethylene levels at an appropriate level, thereby improving plant resistance.

Induction of pathogenic defenses by Methylobaterium has been linked to disease-associated proteins such as β-1,3-glucanase, protective enzymes such as Phenylalanine ammonia-lyase (PAL), and PO (Peroxidase) and PPO ( Related to the production of oxidases such as polyphenyl oxydase. However, the increase or accumulation of activity of these enzymes depends mainly on the plant hormone ethylene. Thus, methyllobacterium with ACC deaminase activity hydrolyzes the ethylene biosynthetic precursor ACC to reduce the stress ethylene levels caused by pathogen infection, while maintaining the level of ethylene necessary for plant growth, resulting in signs of disease. It is known to promote the growth of plants while avoiding. The present invention was carried out to investigate the induction of protective response by controlling the stress ethylene level of tomato exposed to Bacterial spot disease using a methyllobacterium strain producing ACC deaminase.

Meanwhile, Korean Patent No. 0995311 discloses' Phenibacillus polymixsa GBR508 strain having antibacterial activity, nematode impact and plant growth promoting effect and complex disease control and plant growth promoting method using the same. However, as in the present invention, methyllobacterium sp. There is no disclosure about the microbial agent for controlling the control of the bacterium bacillus containing CBMB12 strain as an active ingredient.

The present invention is derived from the above requirements, the inventors of the present invention is a Methylobacterium sp. Xanthomonas campestris pv. Causing bacterial spot disease by inoculation of CBMB12 strain. Bessictoria ( Xanthomonas campestris pv. vesicatoria ) has been shown to promote growth, decrease the incidence of the disease, decrease the amount of ethylene produced and decrease the activity of ethylene biosynthesis-related enzymes. The present invention was completed by confirming that the CBMB12 strain had a control effect against tomato spot bacteria.

In order to solve the above problems, the present invention is methyloacterium ( Methylobacterium sp.) Having a control activity against bacterial spot disease of tomatoes. Provide CBMB12 strain.

In addition, the present invention is the methylobacterium sp. Provided is a composition for controlling antibacterial bacteria of tomato comprising CBMB12 strain or a culture thereof as an active ingredient.

In addition, the present invention is the methylobacterium sp. Provided is a method for controlling tomato bacterium by spraying an effective amount of the CBMB12 strain on a plant or soil.

In addition, the present invention is the methylobacterium sp. It provides a method for producing a composition for controlling anti-bacterial bacterium of tomato comprising culturing the CBMB12 strain.

According to the present invention, the methylobacterium sp. Control of tomato spot bacteria using CBMB12 strain is a natural friendly method without the risk of human toxicity due to environmental pollution and residual pesticides caused by the side effects of pesticides prepared by synthesizing organic compounds. It has the effect of increasing production, and its economic efficiency is high.

1 is a methylbacterium sp. Influence of CBMB12 on stem elongation is shown.
2 is methyllobacterium sp. The inoculation of CBMB12 strain shows the effect of the pathogenesis of tomato infected with spot bacteria. (a) Xanthomonas campestris pv causing spot bacteria. Bessictoria ( Xanthomonas campestris pv. vesicatoria , XCV) inoculated tomato plants; (b) tomato plants inoculated with CBMB12 with XCV; (c) Tomato plants treated with fungicides with XCV.
3 is methyllobacterium sp. The effect of CBMB12 inoculation on the disease index of tomato infected with spot bacteria.
Figure 4 is methyllobacterium sp. Influence of CBMB12 inoculation on the ethylene production of tomato infected with spot bacteria.
5 is methyllobacterium sp. The effect of inoculation of CBMB12 on the accumulation of ACC and ACO in tomato infected with spot bacteria.
6 is methyllobacterium sp. The inoculation of CBMB12 shows the effect of β-1,3-glucanase and PAL expression levels of tomato infected with spot bacteria.
7 is methyllobacterium sp. The effect of inoculation of CBMB12 on the expression of ACO-related genes in tomato infected with spot bacteria.
8 is methyllobacterium sp. The inoculation of CBMB12 shows the effect of β-1,3-glucanase-related gene expression on tomato infected with spot bacteria.
9 is methyllobacterium sp. The effect of inoculation of CBMB12 on the expression of PAL-related genes in tomato infected with spot bacteria.
10 is a fluorescent label methyllobacterium sp. Using a confocal laser microscope. Cluster confirmation of CBMB12 is shown.

In order to achieve the object of the present invention, the present invention is a methylobacterium ( Methylobacterium ) sp. Provide CBMB12 strain. The methylobacterium sp. CBMB12 strain was deposited with the Korean Agricultural Culture Collection (KACC) on February 8, 2012 (Accession No .: KACC 91707P).

In addition, the present invention is methylobacterium sp. Provided is a composition for controlling antibacterial bacteria of tomato comprising CBMB12 strain or a culture thereof as an active ingredient.

In the composition for controlling according to an embodiment of the present invention, the pathogen of the tomato spot bacterium is Xanthomonas campestris fV Besicataria ( Xanthomonas campestris pv. vesicatoria ), but is not limited thereto.

The composition for controlling the antibacterial bacterium of tomato of the present invention may be in the form of a wettable powder or a liquid concentrate. Tomato spot bacteria control composition according to the present invention can be prepared in a liquid form, it may be used in the form of powder powder by adding an extender to it or may be formulated to granulate. However, the formulation is not particularly limited.

Hydrating agent of the present invention is methyl active bacterium sp. CBMB12 strain or culture thereof, white carbon as a hygroscopic agent, sodium bis [2-ethylhexyl] sulfosuccinate as humectant, sodium lignosulfonate as dispersant and kaolin as extender, preferably 10 Wt% methyllobacterium sp. CBMB12 strain or culture thereof, 1 wt% white carbon, 1 wt% sodium bis [2-ethylhexyl] sulfosuccinate, 1 wt% sodium lignosulfonate and 87 wt% kaolin, This is not restrictive.

Liquid hydrating agent of the present invention is methyl active bacterium sp. CBMB12 strains or cultures thereof, wetting agents and dispersants may consist of MBSC (Nonylphenol, ethoxylated, monoether with sulfuric acid, sodium salt, Sodium bis [20 ethylhexyl] sulfosuccinate Polyoxyethylene nonylphenol), isopropanol as an excipient and water as an extender. The liquid hydrating agent is preferably 50% by weight methyllobacterium sp. CBMB12 strain or its culture, 4% by weight MBSC, 30% by weight isopropanol and 16% by weight water, but is not limited thereto.

In addition, the present invention is the methylobacterium ( Methylobacterium ) sp. Provided is a method for controlling tomato bacterium by spraying an effective amount of the CBMB12 strain on a plant or soil.

In the method according to an embodiment of the present invention, the pathogen of the tomato spot bacterial disease may be Xanthomonas campestris fib Besicataria, but is not limited thereto.

An 'effective amount' of the present invention is an amount sufficient to produce a beneficial or desired result, by uniformly diluting the control composition with water in order to control the Xanthomonas Campestris fV Besicataria, and then using a suitable spraying device such as a power sprayer. Can be applied to arable land. When dilution of the hydrating or liquid hydrating agent of the present invention in water, the concentration of the hydrating or liquid hydrating agent is adjusted to about 10 ⁵ to 10 ¹⁰ cfu / ml, preferably 10 ⁸ cfu / ml so that the active ingredient can be in a biologically effective range. May be, but is not limited thereto.

In addition, the present invention is the methylobacterium ( Methylobacterium ) sp. It provides a method for producing a composition for controlling anti-bacterial bacterium of tomato comprising culturing the CBMB12 strain.

Any method known in the art can be used for culturing the strain, and the method is not particularly limited.

In the method according to an embodiment of the present invention, the pathogen of the tomato spot bacterial disease may be Xanthomonas campestris fib Besicataria, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and Methods

Methyl Bacterium Cultivation of Strains and Pathogens

Rice ( Oryza It tumefaciens strain as methyl, which inhabit sativa L.) The method of rice after grinding using the ammonium mineral salts minimal medium (AMS) was added to a 0.5% methanol and carbon Holland Polacco (Plant Physiol 98: 942-948. 1992). 16S rDNA sequences of the isolated methyllobacterium strains were analyzed and identified, and cultured in ammonium mineral salt (AMS) medium to which 0.5% sodium succinate was added for use in the experiment. In addition, Xanthomonas Campestris pv. Besicatoria (XCV) was cultured in nutrient medium (NB), and pathogen strains were distributed by the Department of Botanical Medicine, Chungbuk National University.

Greenhouse experiment

Methylbacterium strains cultured for 72 hours were collected by centrifugation at 10,000 g, 4 ° C for 10 minutes, and then washed twice with 0.03 M MgSO ₄ and then suspended. The concentration of the suspension was adjusted to 1.0 (10 ⁸ cfu ml ⁻¹ ) at OD.600 nm. Tomato used in the experiment ( Lycopersicon esculentum Mill.) Seeds were sterilized by treating 70% ethanol for 1 minute and 2% sodium hypochlorite for 1 minute. Sterilized seeds are washed in sterile water at least 5 times before methylobacterium The cells were inoculated with the strain by stirring for 4 hours in a cell suspension of the strain. The control was stirred using 0.03 M MgSO ₄ .

Seeds inoculated with Methylbacterium strains were seeded in a 50-ball plug seedling tray containing 1: 1 mixed normal and non-soil topsoil. After sowing, the plants were germinated under light / dark conditions at 25 ° C. for 14/10 hours. After germination, the trays were transferred to the greenhouse, and seedlings were inoculated at 5 ml each with a suspension of the Methylbacterium strain. On the 26th day of sowing, the tomatoes were transplanted into a 400 ml plastic pot containing normal horticultural soil and treated with 10 ml Hoagland's culture every week. Each treatment was as follows and each treatment was repeated in eight batches; (1) control, (2) pathogen alone treatment (Xanthomonas Campestris pv. Besicataria), (3) pathogen + Methylbacterium sp. CBMB12, (4) pathogens + fungicides (cosides (spot bacteria control)).

Jantou Monas Campestris pv. Besicataria strain suspension concentrations were OD. Inoculation was inoculated by spraying at 0.4 nm (3 × 10 ⁸ cfu ml ⁻¹ ) at 600 nm on day 3 and day 17 of the transplant. In addition, every week, the suspension of the Methylbacterium strain was OD. Inoculated into the lateral and root zones (10 ml) at a concentration of 1.0 (10 ⁸ cfu ml ⁻¹ ) at 600 nm. Stem elongation of the crops was measured every week, and root length, dry weight of root and stem were measured after harvesting.

Methyl Bacterium Identify the effect of strain inoculation

7 days after inoculation of pathogens, the pathogens were identified and the incidence of disease was measured. The incidence of disease was divided into disease indices of 0-7, as follows; 0-no disease, 1-less than half the leaf area, 2-less than 25% of the diseased leaves, 3-less than 25% of the diseased leaves, 4- Diseased leaves appear from 50% to less than 74%, the same as in steps 5-4, but new young leaves show disease, 6-diseased leaves appear to be 75% or more, 7-crop dies ( Campbell and Madden, Introduction to Plant Disease Epidemiology, John Wiley & Sons . 1990).

Methyl Bacterium  Identifying the Effect of Strain Inoculation on Ethylene Production in Pathogen-Infected Crops

To determine the effect of methylobacterium inoculation on the ethylene production of pathogen-infected crops, the leaves of the crops were sampled. The sampled leaves were placed in a 120 ml GC bottle and blocked for 4 hours by blocking the inlet with a rubber serum stopper. After standing, 1 ml of gas was taken out of the head space and ethylene was measured using gas chromatography (DS 6200, Donam Instruments Inc.). After measuring ethylene, the dry weight of each seedling was measured.

Methyl Bacterium  Inoculation of strains of crops infected with pathogens ACC  Determine impact on stocks

ACC accumulation of plant tissues was measured by the method of Madhaiyan et al. ( Planta 226: 867-876. 2007). 1 g of the plant tissue ground using liquid nitrogen was centrifuged with 5 ml of 80% methanol containing BHT (butylated hydroxytoluene, 2 mg l ^-1 ). The supernatant was concentrated by evaporation in vacuo. ACC concentrations principle of Lizada and Yang (Anal Biochem 100:.. . 142-147 1979) using the method of Wachter (Plant Cell Environ . 22: 1263-1273. 1999). 0.5 ml of an aqueous phase from which dichloromethane was removed was placed in a test tube with 0.1 ml HgCl ₂ (80 mM) and the inlet was closed with a rubber septum. 0.2 ml NaOCl solution was injected into the test tube, mixed, and allowed to stand for 8 minutes. After standing, 1 ml of gas was taken from the headspace and subjected to gas chromatography (DS 6200, Donam Instruments Inc.) to measure oxidized ethylene.

Methyl Bacterium  Inoculation of strains of crops infected with pathogens ACC  Oxidase ( ACO ) Determine impact on activity

ACO (ACC oxidase) activity analysis of plant tissue was performed by crushing frozen plant tissue with liquid nitrogen and then adding 2 ml of extract solution (30 mM sodium ascorbate, 10% (w / v) glycerol to 1 g sample). , 100 mM Tris-HCl (pH 7.2)) was added and then centrifuged at 15,000 rpm for 15 minutes at 4 ° C. After adding 1.5 ml of supernatant, 50 μM FeSO ₄ , 1 mM ACC to the test tube, the inlet was blocked with a rubber septum, and left standing at 30 ° C. for 15 minutes. After standing, 1 ml of gas was taken from the headspace and subjected to gas chromatography (DS 6200, Donam Instruments Inc.) to measure ethylene.

Methyl Bacterium Inoculation of strains of crops infected with pathogens PR  Identify the impact on protein activity

In order to measure the pathogenesis-related protein (PR protein) activity of the plant tissues, the leaves of the sample were ground using liquid nitrogen. After grinding, 1.25 ml of 0.1 M potassium phosphate buffer was added and centrifuged for 20 minutes at 20,000 rpm at 4 ° C. To measure β-1,3-glucanase activity, 50 μl of 50 mM sodium acetate buffer (pH 5.0) added with 10 mg / ml of laminalin was added to 50 μl enzyme extract, followed by mixing at 37 ° C. It was left for an hour. 1.5 ml of dinitrosalicylic acid (dinitrosalicylic acid, DNS) was added thereto, and reacted for 5 minutes in a 100 ° C. constant temperature water bath. The absorbance was measured at 530 nm using an ultraviolet / visible spectrophotometer.

To confirm PAL activity, 1.9 ml of 100 mM Tris-HCl buffer (pH 8.5) and 1 ml of 15 mM L-phenylalanine were added to 100 µl of enzyme extract. After standing at 30 ° C. for 15 minutes, 200 μl of 6 M HCl was added and reacted, and then the absorbance was measured at 290 nm. To measure PO activity, 50 μl of enzyme extract was added with 2.85 ml of 100 mM phosphate buffer (pH 7.0), 50 μl of 20 mM guaical and 20 μl of 40 mM hydrogen peroxide, followed by absorbance at 470 nm. Measured. To measure PPO activity, vortex was added to 200 µl of enzyme extract by adding 1.5 ml of 0.1 M sodium phosphate buffer (pH 6.5) and 200 µl of 0.01 M catechol (pyrocatechol). Thereafter, absorbance was measured using an ultraviolet / visible spectrophotometer at a wavelength of 420 nm.

real time PCR Using ACC -Oxidase, β-1,3- Glucanase  And PAL  Related gene expression measurement

In order to investigate the effect of inoculation of Methylbacterium strains on the expression level of pathogen defense enzymes and related genes in crops, total RNA of plant tissues ground using liquid nitrogen was used for RNeasy plant mini kit (QIAgen, germany). Extracted by. The extracted RNA was quantified using Quant-it (Invitrogen, USA) quantification kit, and treated with DNase I (Invitrogen, USA) to prevent contamination of genomic DNA from the quantified RNA. The synthesis of cDNA was performed using oligo dT of superscript first strand (Invitrogen, USA) together with random hexamer to improve the efficiency of cDNA synthesis. Real-time PCR was analyzed using SYBR green, SYBR green super mix (Bio-rad, USA) and iQ5 (Bio-rad, USA) instrument. CDNA was synthesized using the extracted RNA as a template, and one PCR tube was divided to include cDNA synthesized from 10 ng of total RNA and quantitated using each gene specific primer. Quantification used a standardized relative quantitative method using actin and tubulin as internal controls.

In the crop GFP  Transformation Methyllobacterium Cluster  Confirm

In order to visually identify the inoculation effect of the methylobacterium strain, the fluorescent label using the genetic engineering technique and the community of microorganisms in the crop were identified. The population of GFP transformed methyllobacterium in the crop was analyzed using a Confocal laser scanning microscope (CLSM). The collected leaves were cut to about 1 cm ² and placed on a slide glass, and treated with Vectashield mounting medium (Vector Labs, Burlingame, CA), and then covered with a cover glass. Microscopy was performed using a Leica TCS SP2 confocal system (Leica Microsystems Heidelberg GmbH, Manheim, Germany) and an argon ion laser (GFP: excitation, 488 nm; emission filter BP 500-530), CLSM Fluorescently labeled strains were identified using the system (version 2.5.1227a) software.

Example  One. Methyl Bacterium Effect of Strain Inoculation on Plant Growth

On the 26th day of sowing, the tomatoes were transplanted into a 400 ml plastic pot containing general garden soil, and then the Xanthomonas campestris pv. Bessictoria ( Xanthomonas campestris pv. vesicatoria, XCV) strain suspension is OD. Inoculation was inoculated by spraying on the 3rd and 17th day of transplantation at a concentration of 0.4 (3 × 10 ⁸ cfu ml ⁻¹ ) at 600 nm. Methylbacterium strain suspensions are also OD. Inoculated into the lateral and root zones (10 ml) each week at a concentration of 1.0 (10 ⁸ cfu ml ⁻¹ ) at 600 nm. Stem elongation was measured every week for untreated control plants, tomato plants inoculated with XCV causing spot bacillus, tomato plants inoculated with CBMB12 with XCV and tomato plants treated with fungicide with XCV. Compared to the control and spot bacteria pathogens single inoculation treatment, the tomato exposed to the spot bacteria disease, Methylbacterium sp. In the treatment group inoculated with CBMB12 strain it was confirmed that the stem height promotion of the crop (Fig. 1), the significant increase in the root length, ground dry weight and root dry weight of the crop (Table 1).

Methylbacterium sp. Effect of CBMB12 Inoculation on Root Length, Ground Dry Weight and Root Dry Weight of Crops process Root Length (cm / plant) Stem Dry Weight (g / plant) Root Dry Weight (g / plant) Control 24.0 ± 1.5b 10.0 ± 0.3b 0.45 ± 0.01 b XCV 19.9 ± 1.7c 8.2 ± 1.0c 0.39 ± 0.04b XCV + CBMB12 29.5 ± 1.6a 12.3 ± 0.7a 0.58 ± 0.06a XCV + Sterilizer 31.6 ± 2.0a 9.7 ± 0.4bc 0.43 ± 0.01 b

Values with identical letters (mean ± standard error, n = 8) are not significant at 0.05% (LSD).

Example  2. Methyl Bacterium Effect of Strain Inoculation on Disease Development

Seven days after the inoculation of the bacteriophage pathogen, the pathology was confirmed by the pathogen and the incidence of the disease was divided by the disease index. Disease index shows no disease (0), less than half of leaf area (1), less than 25% of diseased leaves (2), 25% or less than 49% of diseased leaves (3), the diseased leaves appear to be less than 50% to 74% (4), the same as step 4, but the new young leaves show the disease (5), the diseased leaves appear to be more than 75% (6), crops were divided into eight stages of death (7) and measured at 14, 21, 28, and 35 days of inoculation of pathogens. The pathology and disease index of the leaves of tomato infected with spot bacterium were measured. Methylbacterium sp. Inoculation of CBMB12 strain showed significantly lower pathology and a significant reduction in disease index compared to the pathogen single inoculation treatment (FIGS. 2 and 3).

Example  3. Methyl Bacterium  Strain inoculation ACC , ACO , β-1,3- Glucanase  And effect on PAL expression

As a result of confirming the occurrence of stress level ethylene caused by S. aureus bacterial infection and the accumulation of ACC and ACO (ACC oxidase) involved in tomato, CBMB12 inoculation treatment showed a significant reduction in ethylene generation, ACC and ACO accumulation in tomato also confirmed a significant decrease (Figs. 4, 5). This is because ACC accumulated in crops was hydrolyzed to α-ketobutyrate and ammonia by ACC deaminase produced by methyllobacterium.

In addition, the results of confirming the expression of β-1,3-glucanase and PAL, which are pathogen-protecting enzymes of crops, also showed the effect of methyllobacterium sp. CBMB12 Significant increase was confirmed in the strain inoculation treatment (FIG. 6).

In addition, ACO, an enzyme involved in ethylene production in crops, and β-1,3-glucanase, a pathogen-protective enzyme, and PAL production-related gene expression were identified using RT-PCR. As a result, methylobacterium sp. Significant decrease in ACO-related gene expression was observed in the CBMB12 strain treatment (FIG. 7). In addition, in the case of β-1,3-glucanase and PAL production-related gene expression, methyllobacterium sp. The gene expression was higher than that of the CBMB12 strain untreated control (Figs. 8 and 9).

Example  4. GFP  Transformation Methyllobacterium Cluster

In order to visually confirm the inoculation effect of the Methylbacterium strain, Methylbacterium was transformed with GFP to confirm the colonization of microorganisms in the crop using fluorescent labels. As a result of analyzing the fluorescent label of the colony of GFP-transformed methyllobacterium using CLSM, Methylbacterium sp. As the CBMB12 strain was clustered in the tomato plant, it was confirmed that the result of controlling the stress ethylene level and improving the crop resistance (FIG. 10).

Agricultural Biotechnology Research Institute KACC91707 20120208

Claims (7)

Methylobacterium sp., Which is active against bacterial spot disease of tomatoes. CBMB12 strain (KACC 91707P). A composition for controlling anti-bacterial bacterium of tomato comprising the strain of claim 1 or a culture thereof as an active ingredient. According to claim 2, wherein the pathogen of the tomato spot bacterial disease Xanthomonas Campestris fV Becciatoria ( Xanthomonas campestris pv. vesicatoria ) composition for controlling. Methylobacterium sp. A method for controlling tomato bacterium by spraying an effective amount of CBMB12 strain on a plant or soil. 5. The method according to claim 4, wherein the pathogen of tomato spot bacterium is Xanthomonas Campestris fib Besicataria. Methylobacterium sp. Method for producing a composition for controlling the bacteriostatic bacterium of tomato comprising culturing CBMB12 strain. 7. The method according to claim 6, wherein the pathogen of tomato spot bacterium is Xanthomonas campestris fib Besicataria.

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