CN105349453A - Serratia nematodiphila strain and application thereof - Google Patents
Serratia nematodiphila strain and application thereof Download PDFInfo
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Abstract
The invention discloses a Serratia nematodiphila strain and application thereof. The strain is classified and named as Serratia nematodiphila PR4, collected at the China General Microbiological Culture Collection Center, in Number 3 of Number 1 Yard on the Beichen west road in the Chaoyang District of Beijing, on 28th, August, 2014 and numbered as CGMCC No.9614. The strain has good degrading effect on protein. Enzyme activity, of protease generated by the strain, measured by a Folin-phenol method is 3.03 IU. The strain has good phosphorus dissolving effect measured by using a molybdenum blue spectrophotometry method. It shows that the PR4 strain has potential application value in promoting tobacco growth.
Description
Technical field
The invention belongs to plant growth-promoting rhizobacteria and applied technical field thereof, be specifically related to the application addicted to nematode Serratia and growth-promoting characteristic thereof with degrade proteins and phosphorus decomposing function that a strain promotes tobacco growing.
Background technology
Tobacco is Solanaceae (Solanaceae) Nicotiana (Nicotiana) annual herb plant.Happiness temperature, happiness light, drought-enduring, be afraid of flood, resistance to lean, need potassium more.Tobacco leaf, stem, calyx and fruit have cellulous hair, wherein contain chlorophyll in glandular hairs cell, comprehensively containing exudate that is gummy and resin, can have certain relation with the fragrance of tobacco.Tobacco petiole is not obvious or become aliform handle, and its panicle top is raw, calyx tubular, corolla funnel-form, end pink.Capsule, seed tawny.Tobacco originates in South America, because its blade is containing nicotine (Nicotine), through overmodulation, classification and processing treatment after gathering, can cigarette processed, cigar, tobacco etc., and be the important raw material of tobacco industry.There is very large problem in China's tobacco field fertilizing in recent years, is embodied in: (1) utilization rate of fertilizer is not high, and China's flue-cured tobacco utilization rate of fertilizer is lower to have data to show, some province utilization rate of fertilizer of south only about 20%.Its reason lacks further investigation to the correlation technique of utilization rate of fertilizer, particularly fundamentally do not research and solve the control of Main Tobacco-growing Regions In South fertilizer loss and the nutrient malabsorption etc. in northern cigarette district; (2) there is downtrending in various degree in vega soil fertility, and owing to lacking good culture fertility measure, crop rotation condition is in addition poor, the long-term continuous cropping of vega, and soil main nutrient elements occurs downtrending all in various degree, and soil physical property becomes bad simultaneously; (3) nutritional imbalance, the light phosphorus potassium deficiency of diazonium; (4) research is lacked to the fertilizer practice of dry farming tobacco.
Research finds can the microorganism of Promoting plant growth containing some in plant rhizosphere soil, generally has fixed nitrogen, phosphorus decomposing, releases potassium, produces the ability such as plant hormone and secretion microbiotic or at least have the bacterium, cyanobacteria etc. of one of them ability.Be referred to as plant growth-promoting rhizobacteria (PGPR), its growth-promoting functions mechanism mainly contains following several large class: provide plant nutrient substance, produce plant growth regulating substance, plant rhizosphere bioremediation agents and environment-stress conditioning agent etc.But filter out and there is several functions and efficiently microorganism strains is the target that this area is pursued always.
Proteolytic enzyme is the class of enzymes of catalytic proteins hydrolysis.The diversity of protease species and the specificity of hydrolytic activity, make it become the industrial enzyme with important commercial value at field of industrial productions such as food, leather, washing composition.Containing large amount of organic in soil, PGPR can be utilized to produce proteolytic enzyme by the nitrogenous organic substance decomposing in soil, produce and can be used for the nitrogen of Tobacco, so just can reduce the use of chemical fertilizer, avoid the destruction to soil and environment.The degraded of present report is keratic bacillus thuringiensis (Bacillusthuringiensis), degrading ossein albumen have active micrococci (Micrococcusaqilis).But yet there are no have simultaneously efficient degradation protein and phosphorus decomposing function addicted to nematode Serratia and the application report promoting tobacco growing thereof.
Summary of the invention
The object of this invention is to provide a strain addicted to nematode Serratia and the application promoting tobacco growing thereof, this bacterial strain has degrade proteins and phosphorus decomposing function.
One strain addicted to nematode Serratia (Serratianematodiphila) PR4, this bacterial strain deposit number: CGMCCNo.9614.Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on August 28th, 2014.
The described optimal culture conditions addicted to nematode Serratia, the LB substratum of employing: peptone 10g, yeast extract 5g, NaCl10g, distilled water 1000ml, 121 DEG C of sterilizing 20min, culture temperature 37 DEG C, pH7.0.
The described application addicted to nematode Serratia, for degrade proteins and/or phosphorus decomposing, thus promotes the growth of plant.Described plant comprises tobacco.
Obtain bacteria suspension after strain culturing of the present invention to use as liquid microbial inoculum, bacterium Particle density cfu>=10 of bacteria suspension
8individual/mL.The using method of microbial inoculum: fill with root after slow seedling, every strain cigarette seedling is with microbial inoculum 1mL.
Bacterium colony and the thalline of bacterial strain of the present invention are characterized as: this bacterial strain is at LB substratum (yeast extract 5g, peptone 10g, sodium-chlor 10g, distilled water 1000mL, agar 15 ~ 20g, 121 DEG C of sterilizing 20min, pH7.0,37 DEG C) upper cultivate 48h after bacterium colony rounded, opaque, smooth surface is moistening, oyster white, neat in edge, bacterium colony is slightly protruding.Thalline is shaft-like, produces gemma, Gram-negative.
The physiological and biochemical property of this bacterial strain is: methyl red test is negative, v-p tests the positive, Starch Hydrolysis negative, nitrate reduction test is positive, indole test is negative, hydrogen sulfide production test is positive, Citrate trianion utilizes test positive, glucose fermentation test is positive, sucrose fermentation test is positive, maltose fermentation test is positive, lactose-fermentation test is positive, mannose ferment test is positive, glucose carbon source utilizes test positive, the test of lactose utilization of carbon source is positive, the test of N.F,USP MANNITOL utilization of carbon source is positive, the test of sucrose utilization of carbon source is positive, the test of maltose utilization of carbon source is positive, citric acid utilization of carbon source negative, 2 ﹪ NaCl Salt tolerances are positive, 5 ﹪ NaCl Salt tolerances are positive, 7 ﹪ NaCl Salt tolerances are positive, 10 ﹪ NaCl Salt tolerances are positive, product ammonia test is positive.
The optimal culture conditions of this bacterial strain is: LB substratum: peptone 10g, yeast extract 5g, NaCl10g, distilled water 1000ml, temperature 37 DEG C, pH7.0.
This bacterial strain may be used for preparing microbiobacterial agent; Bacteria suspension (bacterium Particle density cfu>=10 of bacteria suspension are obtained by LB culture medium culturing during application
8individual/mL), as liquid microbial inoculum.
The using method of microbial inoculum: fill with root after slow seedling, every strain cigarette seedling is with microbial inoculum 1mL.
As follows to the functional examination result of bacterial strain of the present invention: at substratum (the skim-milk 3.0g of skim-milk, agar 3.0g, deionized water 200mL, pH7.0 ~ 7.2,108 DEG C of sterilizing 20min) in cultivate 24h (37 DEG C) after to record its colony radius be 0.2cm, transparent circle radius is 1.4cm, and transparent circle radius is 7 with the ratio of colony radius; Be that substrate records the proteinase activity of PR4 for 3.03IU by National Standard Method with casein.At Meng Jinna solid medium (glucose 10g, (NH
4)
2sO
40.5g, NaCl0.3g, MgSO
47H
2o0.3g, FeSO
40.03g, MnSO
4h
2o0.03g; CaCO
35.0g, KCl0.3g, Ca
3(PO
4)
225.0g, Yelkin TTS 0.3g, agar 18 ~ 20g, pH value 7.2 ~ 7.4) go up cultivation one week, recording its colony diameter of first day d is 2mm, and molten phosphorus loop diameter D is 5mm, D/d is 2.5, within 3rd day, its colony diameter d is 3mm, molten phosphorus loop diameter D is 12.5mm, D/d be the 4.2, seven day its colony diameter d is 3mm, molten phosphorus loop diameter D is 18mm, D/d is 6; At inorganic phosphorus liquid nutrient medium (C
6h
12o
610.0g, (NH
4)
2s0
40.5g, NaCl0.3g, KCl0.3g, MgS0
47H
2o0.3g, FeS0
47H
2o0.03g, MnS0
44H
2o0.03g, Ca
3(PO
4)
225.0g, water 1000mL, pH7.0-7.5.) cultivate after 4 days, the content of rapid available phosphorus in nutrient solution is measured with molybdenum blue colorimetric method, thus the dissolving P capacity of reacting bacteria, recording available phosphorus contents in PR4 nutrient solution is 42.4mg/L.Through the cigarette strain number of blade of PR4 process when potted plant 30 days, maximum blade surface-area is all greater than contrast, but do not reach significant difference, through the cigarette strain stem girth of PR4 process when 60 days, the number of blade is all higher than contrast, and plant height and surface-area are a little less than contrast, stem girth reaches conspicuous level in 0.05 level, contrast is better than through the cigarette strain plant height of PR4 process and the number of blade when 90 days, through the root of the tobacco plant of PR4 process, stem, leaf, the total fresh weight of plant is all better than control treatment, but only have root to reach significant difference, to the root can finding out plant in biological quantitative statistics after gathering, stem, leaf dry weight and plant gross dry weight are all better than contrast, compared with the control, root dry weight amplification is 26.58%, the heavy amplification of stem is 16.83%, leaf dry weight amplification is 2.53%, plant gross dry weight amplification is 7.86%.Above result of study shows, PR4 bacterial strain can well promote tobacco growing, in promotion tobacco growing, have potential using value.
The present invention is addicted to nematode Serratia (Serratianematodiphila) PR4, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on August 28th, 2014, deposit number: CGMCCNo.9614.
Accompanying drawing explanation
Fig. 1 is 60 days and PR4 process in 90 days and the cigarette strain plant height column diagram contrasted;
Fig. 2 is 60 days and PR4 process in 90 days and the cigarette strain stem girth column diagram contrasted;
Fig. 3 is that 30 days, 60 days and PR4 process in 90 days amass column diagram with the maximum leaf surface of cigarette strain contrasted;
Fig. 4 is 30 days, 60 days and PR4 process in 90 days and the cigarette strain number of blade column diagram contrasted;
Fig. 5 is PR4 process biomass column diagram fresh in the cigarette strain contrasted;
Fig. 6 is PR4 process and the cigarette strain dry biomass column diagram contrasted;
Fig. 7 PR4 degrade proteins design sketch;
Fig. 8 PR4 phosphorus decomposing design sketch.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The present invention is addicted to nematode Serratia (Serratianematodiphila) PR4, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on August 28th, 2014, deposit number: CGMCCNo.9614.
(1) screening of CGMCCNo.9614
Process in the enrichment of beef extract-peptone liquid nutrient medium: get gather from level ground, Sangzhi, Hunan land owned by officials, Liuyang cigarette section tobacco rhizosphere soil, totally 10 samples, be numbered G1, G2, G3, G4, G5, L1, L2, L3, L4, L5, each soil sample takes 10g, add in 100mL beef extract-peptone liquid nutrient medium, 191r/min, 37 DEG C of shaking table shaking culture 24h.
Take skim-milk as the process of degradation of substrates protein: get the nutrient solution after enrichment after gradient dilution, from 10
-1, 10
-2, 10
-3, 10
-4drawing 100 μ L diluents in extent of dilution coats on Gause I substratum, cultivates 3 days under 28 DEG C of conditions; From 10
-3, 10
-4, 10
-5, 10
-6drawing 100 μ L in extent of dilution is coated on PDA substratum, cultivates 1 day under 37 DEG C of conditions; From 10
-5, 10
-6, 10
-7, 10
-8drawing 100 μ L in extent of dilution is coated on beef-protein medium, cultivates 1 day under 37 DEG C of conditions.
With sterilizing toothpick, bacterium colony is chosen on skim-milk substratum after flat board to be coated growing bacterium.Observe and choose the bacterial isolates that can produce obvious hydrolysis, after so repeatedly verifying repeatedly, picking can be stablized the bacterial strain producing transparent circle and carry out purifying repeatedly, each dull and stereotyped dibbling 4 strain bacterial strain on skim-milk substratum again, 37 DEG C of constant temperature culture, measure transparent circle and the colony diameter of different strains after 72h.According to the size of transparent circle with colony radius ratio (HC) value, the relatively strong bacterial strain of proteolytic enzyme ability is produced in screening.By the colony inoculation after purifying to (yeast extract 5g, peptone 10g, sodium-chlor 10g, distilled water 1000mL, agar 15 ~ 20g, 121 DEG C of sterilizing 20min, pH7.0 in LB solid slant culture base.) preserve.Obtaining 144 strains altogether can the bacterial strain of degrade proteins.
By dull and stereotyped transparent circle method, GB Protease assays, ultraviolet spectrophotometry surveys growth hormone content, protect green method, ability of dissolving potassium assay method, molten phosphorus circle method and molybdenum blue colorimetric method measure the ability of its degrade proteins respectively, produce ability, ability of dissolving potassium and the phosphate solubilization of phytokinin.Filter out a strain can have again the bacterial strain of phosphorus decomposing effect by degrade proteins from being separated the bacterial strain that obtains, be designated as PR4.
(2) CGMCCNo.9614 strain identification
The extraction of genomic dna: will be separated after the bacterial strain streak inoculation that obtains cultivates 24h to LB solid medium in 37 DEG C, picking list bacterium colony in the test tube that LB liquid nutrient medium is housed 37 DEG C, 12h cultivated by 150r/min shaking table.Concrete grammar consults document (AusubelFM, BrentR, KingstonRE, etal.ShortProtocolsinMolecularBiology.Chichester:JohnWil ey & Sons, Inc, 1995:36-40.).The DNA obtained is dissolved in 50 μ LTE damping fluids (10mMTris-HCl, 1mMEDTA, PH=8.0), is then stored in-20 DEG C of refrigerators for subsequent use.
16SrDNA sequence amplification primer is synthesized by Shanghai biotechnology company limited.
Pcr amplification reaction system: Taq DNA polymerase 0.5 μ L, 10 × Buffer5 μ L, MgCl
23 μ L, dNTPs (10mmol/L) 1 μ L, primer 2 7F1 μ L, primer 1492R1 μ L, template 0.5 μ L, ddH
2o complements to 50 μ L.Reaction conditions: 95 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1.5min, 30 circulations; 72 DEG C extend 10min.The examining order of 16SrDNA is completed by Shanghai biotechnology company limited.
By the sequence alignment in surveyed 16SrDNA sequence and GenBank database, result shows: bacterial strain of the present invention is arranged in phylogenetic tree Serratia (Serratia.sp) branch, have nearest sibship with Serratianematodiphila (EU914257.1) and Serratiamarcescens (AB244453.1) two strain bacterium, its 16SrRNA gene order similarity is all 99%.
This bacterial strain is at LB substratum (yeast extract 5g, peptone 10g, sodium-chlor 10g, distilled water 1000mL, agar 15 ~ 20g, 121 DEG C of sterilizing 20min, pH7.0,37 DEG C) upper cultivate 48h after bacterium colony rounded, opaque, smooth surface is moistening, oyster white, neat in edge, and bacterium colony is slightly protruding.Thalline is shaft-like, produces gemma, Gram-negative.The physiological and biochemical property of this bacterial strain is: methyl red test is negative, v-p tests the positive, Starch Hydrolysis negative, nitrate reduction test is positive, indole test is negative, hydrogen sulfide production test is positive, Citrate trianion utilizes test positive, glucose fermentation test is positive, sucrose fermentation test is positive, maltose fermentation test is positive, lactose-fermentation test is positive, mannose ferment test is positive, glucose carbon source utilizes test positive, the test of lactose utilization of carbon source is positive, the test of N.F,USP MANNITOL utilization of carbon source is positive, the test of sucrose utilization of carbon source is positive, the test of maltose utilization of carbon source is positive, citric acid utilization of carbon source negative, 2 ﹪ NaCl Salt tolerances are positive, 5 ﹪ NaCl Salt tolerances are positive, 7 ﹪ NaCl Salt tolerances are positive, 10 ﹪ NaCl Salt tolerances are positive, product ammonia test is positive.By above bacterium colony, thalli morphology, physiological and biochemical property and 16SrDNA sequential analysis, identify that CGMCC9614 is addicted to nematode Serratia (Serratianematodiphila).
(3) proteolytic degradation test
Substratum and bacterial classification preparation: substratum (the skim-milk 3.0g of preparation skim-milk; Agar 3.0g; Deionized water 200mL; PH7.0 ~ 7.2,108 DEG C of sterilizing 20min).After being down flat plate, this bacterium shaking table in plate culture medium is cultivated 24h (37 DEG C).Observe transparent circle size and measure transparent circle radius (R) and colony radius (r), and measure its ratio R/r.Be 0.2cm through measuring its colony radius, transparent circle radius is 1.4cm, R/r=7.
National Standard Method surveys proteinase activity power: National Standard Method surveys proteinase activity power: with Forint phenol method, is defined as an enzyme (IU) alive with casein for the every 1min of substrate generates enzyme amount needed for 1 μ g tyrosine.The proteinase activity of PR4 is 3.03IU after measured.
(4) dissolving P capacity measures
The present invention is screened the inoculation that obtains at Meng Jinna solid medium (glucose 10g, (NH
4)
2sO
40.5g, NaCl0.3g, MgSO
47H
2o0.3g, FeSO
40.03g, MnSO
4h
2o0.03g; CaCO
35.0g, KCl0.3g, Ca
3(PO
4)
225.0g, Yelkin TTS 0.3g, agar 18 ~ 20g, pH value 7.2 ~ 7.4) go up cultivation one week, measure its colony diameter d, molten phosphorus loop diameter D, calculate D/d.As shown in Figure 8, record its colony diameter of first day d is 2mm to phosphorus decomposing circle effect, and molten phosphorus loop diameter D is 5mm, D/d is 2.5, within 3rd day, its colony diameter d is 3mm, and molten phosphorus loop diameter D is 12.5mm, D/d is 4.2, within 7th day, its colony diameter d is 3mm, and molten phosphorus loop diameter D is 18mm, D/d is 6.By PR4 at inorganic phosphorus liquid nutrient medium (glucose 10.0g, (NH
4)
2sO
40.5g, NaCl0.3g, KCl0.3g, MgS0
47H
2o0.3g, FeSO
47H
2o0.03g, MnSO
44H
2o0.03g, Ca
3(PO
4)
225.0g, water 1000mL, pH7.0-7.5.) middle cultivation is after 4 days, molybdenum blue colorimetric method measures the content of rapid available phosphorus in nutrient solution, thus the dissolving P capacity of reacting bacteria.Recording available phosphorus contents in PR4 nutrient solution is 42.4mg/L.
(5) pot experiment
First be nursery, select the tobacco seed of same kind to carry out nursery.Stalwartness is selected in nursery for 30 days afterwards, the cigarette seedling that anosis, growing way is consistent is transplanted, and the strain of every potted plant cigarette 1, mainly all imbeds cigarette seedling cane in soil, water the water of equivalent after transplanting immediately.
Then be process, one group of blank, one group adds PR4 microbial inoculum.Cultivate to obtain bacteria suspension of the present invention (bacterium Particle density>=10 of bacteria suspension
8cfu/mL), use as liquid microbial inoculum, fill with root after slow seedling, every strain cigarette seedling is with microbial inoculum 1mL.
Pendulum basin: for reducing because the difference of booth different directions temperature is on the impact of experimental result, basin should be put along with booth vertical direction.
Soil humidity is noted in the tobacco growing phase, then need moisturizing to 80% (moisturizing can be determined according to the growth of cigarette strain and lack of water situation, but must ensure that the irrigation amount of every basin and soil humidity are consistent) of maximum water holding capacity lower than after soil relative water content 50%.
Every 10 days investigation numbers of blade after transplanting, plant height, stem girth, leaf look, maximum leaf length and width, growing way, residing breeding time etc.).When finding that between process, growing way appearance has a notable difference in potted plant process, contrast is taken pictures together with process pendulum, the root system sampling cigarette strain is carried out contrast simultaneously and take pictures.
Result is as shown in table 1, table 2, table 3, table 4, table 5.
Table 1.30 day PR4 is on the impact of tobacco agronomy character
Note: data bit mean number ± standard deviation in table.The significance of difference (p<0.05) is shown with lowercase alphabet different after column data, lower same.
Table 2.60 day PR4 is on the impact of tobacco agronomy character
Table 3.90 day PR4 is on the impact of tobacco agronomy character
Table 4.PR4 is on the impact of cigarette strain fresh weight
Table 5.PR4 is on the impact of cigarette strain dry weight
In conjunction with the accompanying drawings and embodiments the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (8)
1. a strain is addicted to nematode Serratia (Serratianematodiphila) PR4, this bacterial strain deposit number: CGMCCNo.9614.
2. the culture condition addicted to nematode Serratia according to claim 1, is characterized in that, the LB substratum of employing: peptone 10g, yeast extract 5g, NaCl10g, distilled water 1000ml, 121 DEG C of sterilizing 20min, culture temperature 37 DEG C, pH7.0.
3. described in claim 1 addicted to the application of nematode Serratia, it is characterized in that, for degrade proteins and/or phosphorus decomposing.
4. the application addicted to nematode Serratia according to claim 3, is characterized in that, for promoting the growth of plant after degrade proteins and/or phosphorus decomposing.
5. the application addicted to nematode Serratia according to claim 4, is characterized in that, described plant comprises tobacco.
6. the application addicted to nematode Serratia according to claim 3 or 4 or 5, is characterized in that, obtains bacteria suspension and use as liquid microbial inoculum after strain culturing.
7. the application addicted to nematode Serratia according to claim 6, is characterized in that, bacterium Particle density>=10 of bacteria suspension
8cfu/mL.
8. the application addicted to nematode Serratia according to claim 7, is characterized in that, the using method of microbial inoculum: fill with root after slow seedling, every strain cigarette seedling is with microbial inoculum 1mL.
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| CN116814491A (en) * | 2023-06-29 | 2023-09-29 | 四川农业大学 | Serratia MYH_7 and application thereof |
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| CN116904366B (en) * | 2023-08-04 | 2024-06-25 | 西南大学 | Serratia nematophila, microorganism coating preparation and application thereof |
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| CN114058562A (en) * | 2021-11-19 | 2022-02-18 | 杭州师范大学 | Recombinant Serratia expressing dsRNA and application thereof |
| CN114058562B (en) * | 2021-11-19 | 2023-08-18 | 杭州师范大学 | Recombinant Serratia expressing dsRNA and application thereof |
| CN116814491A (en) * | 2023-06-29 | 2023-09-29 | 四川农业大学 | Serratia MYH_7 and application thereof |
| CN116814491B (en) * | 2023-06-29 | 2024-01-26 | 四川农业大学 | Serratia MYH_7 and application thereof |
| CN116904366A (en) * | 2023-08-04 | 2023-10-20 | 西南大学 | Serratia nematophila, microorganism coating preparation and application thereof |
| CN116904366B (en) * | 2023-08-04 | 2024-06-25 | 西南大学 | Serratia nematophila, microorganism coating preparation and application thereof |
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