CN105255769B - One Enterobacter cloacae and its application - Google Patents
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Abstract
The invention discloses an Enterobacter cloacae and its applications.The strain classification is named as enterobacter cloacae (Enterobacter cloacae) JP6, depositary institution:China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date:On August 28th, 2014, deposit number:CGMCC No.9615.Bacterial strain of the present invention has good phosphorus decomposing effect.Ultraviolet spectrophotometry measures bacterial strain production auxin.Show JP6 bacterial strains to promoting tobacco growing to have potential application.
Description
Technical field
The invention belongs to plant growth-promoting rhizobacteria and its applied technical fields, and in particular to the tool of one plant of promotion tobacco growing
There is phosphorus decomposing and produces the enterobacter cloacae of IAA functions and its application of growth-promoting characteristic.
Background technology
Tobacco is Solanaceae (Solanaceae) Nicotiana (Nicotiana) annual herb plant.Happiness temperature, light, it is drought-enduring,
Be afraid of flood, it is resistance to it is lean, need potassium more.Tobacco Leaf, stem, calyx and fruit have the hair of many cells, wherein contain chlorophyll in glandular hairs cell,
Can be comprehensive containing gummy and resin exudate, there is certain relationship with the fragrance of tobacco.Tobacco petiole unobvious or at aliform handle,
Its panicle basidixed, calyx tubular, corolla funnel-form, end pink.Capsule, seed yellowish-brown.Tobacco originates in South America
It continent, can cigarette processed, cigar, tobacco through ovennodulation, classification and working process after harvesting because of its blade (nicotine) containing nicotine
Deng being the important raw material of tobacco industry.There is prodigious problem in China's tobacco field fertilizing in recent years, is embodied in:(1) fertilizer profit
It is not high with rate, data show that China's flue-cured tobacco utilization rate of fertilizer is relatively low, some southern province utilization rate of fertilizer only 20% or so.Its
The reason is that the correlation technique to utilization rate of fertilizer lacks further investigation, the especially control to Main Tobacco-growing Regions In South fertilizer loss and north
Nutrient malabsorption of square cigarette district etc. is not researched and solved fundamentally;(2) vega soil fertility, which occurs declining in various degree, becomes
Gesture, due to lacking good culture fertility measure, crop rotation condition is poor in addition, and the long-term continuous cropping of vega, soil main nutrient elements is all
There is downward trend in various degree, while soil physical property deteriorates;(3) nutritional imbalance, the light phosphorus potassium deficiency of diazonium;(4) to drought
The fertilizer practice for making tobacco lacks research.
Research finds that the microorganism of plant growth can be promoted in plant rhizosphere soil containing some generally have fixed nitrogen, solution
Phosphorus, Potassium release generate plant hormone and secrete the abilities such as antibiotic or at least have the bacterium, cyanobacteria etc. of one of them ability.
It is referred to as plant growth-promoting rhizobacteria (PGPR), growth-promoting functions mechanism mainly there are following a few major class:Plant nutrient substance, production are provided
Plant growth regulatory substance, plant rhizosphere bioremediation agents and environment-stress conditioning agent etc..But it filters out with multiple functions
And efficient microbial strains are always the target that this field is pursued.
Tobacco absorbs a great number of elements nitrogen, phosphorus, potassium, moderate-element calcium, magnesium, sulphur and micronutrient boron, zinc, copper, manganese, iron etc.
Nutrient.Absorbing rule is that early growth period (after transplanting to group before) is few, and mid-term (group is to before pinching) uptake is most, after
Phase (after topping) uptake gradually decreases, the entire S-type curve of growth period nutrient absorption.Existing phosphorus can not meet in soil
The needs of tobacco growing.Contain large amount of organic in soil, PGPR can be utilized to decompose the phosphorous organic matter in soil, production
Life can supply the phosphorus of Tobacco.Both it had been the drawbacks of tobacco in turn avoids excessively applying chemical fertilizer supplemented with nutrient,
For example it is easy pollution environment, so that the soil property that soil hardening makes Tobacco Farm is destroyed, the organic matter in soil is lost, reduces soil
Earth fertility etc..And it yet there are no while having efficient phosphate-solubilizing and produce the enterobacter cloacae of IAA functions and its promote tobacco life
Long application report.
Invention content
The object of the present invention is to provide the application of an Enterobacter cloacae and its promotion tobacco growing, which has phosphorus decomposing
With the effect of production auxin IAA.
One Enterobacter cloacae (Enterobacter cloacae) JP6, the bacterial strain deposit number:CGMCC No.9615.
Depositary institution:China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address:Chaoyang District, Beijing City
The institute 3 of North Star West Road 1, preservation date:On August 28th, 2014.
The condition of culture of the enterobacter cloacae, using LB culture mediums:Peptone 10g, yeast extract 5g, NaCl
10g distills 1000ml, 121 DEG C of sterilizing 20min, 37 DEG C of cultivation temperature, pH 7.0.
The application of the enterobacter cloacae, for phosphorus decomposing and/or production auxin IAA, to promote the growth of plant.Institute
The plant stated includes tobacco.
It obtains bacteria suspension after strain culturing of the present invention to use as liquid microbial inoculum, bacteria concentration >=10 of bacteria suspension8cfu/
mL.The application method of microbial inoculum:Pouring root after slow seedling, every plant of tobacco seedlings add microbial inoculum 1mL.
The bacterium colony and thalline of bacterial strain of the present invention are characterized as:The bacterial strain is in LB culture mediums (yeast extract 5g, peptone 10g, chlorine
Change sodium 10g, distilled water 1000mL, agar 15~20g, 121 DEG C of sterilizings 20min, pH7.0,37 DEG C) on after culture 48h bacterium colony be in
Circle, opaque, dry tack free is without protuberance, faint yellow, neat in edge.Thalline is in spherical, does not produce gemma, and Gram's staining is cloudy
Property.
The physiological and biochemical property of the bacterial strain is:Methyl red test feminine gender, v-p negatives, Starch Hydrolysis the experiment positive, nitre
Hydrochlorate reduction test is positive, indole test is positive, hydrogen sulfide production test is positive, citrate utilizes and tests positive, glucose fermentation
Negative, sucrose fermentation test is negative, malt sugar fermentating test is negative, lactose-fermentation test is positive, mannose ferment experiment
Negative, glucose carbon source is positive using experiment, the experiment of lactose utilization of carbon source is positive, mannitol utilization of carbon source tests positive, sucrose
Utilization of carbon source experiment is positive, maltose utilization of carbon source tests the positive, citric acid utilization of carbon source negative, 2 ﹪ NaCl salt tolerants try
Test positive positive, the 5 ﹪ NaCl Salt tolerances positives, 7 ﹪ NaCl Salt tolerances, 10 ﹪ NaCl Salt tolerances feminine genders, production ammonia test
It is positive.
The optimal culture conditions of the bacterial strain are:Yeast extract 5g, peptone 10g, sodium chloride 10g, distilled water 1000mL, fine jade
Fat 15~20g, pH7.0,37 DEG C of temperature.
The above bacterial strain can be used for preparing microbial bacterial agent, bacteria suspension be obtained with LB medium cultures, as liquid microbial inoculum.
Bacteria concentration >=10 of bacteria suspension8cfu/mL。
The application method of microbial inoculum:Every plant of tobacco seedlings of pouring root add microbial inoculum 1mL after slow seedling.
Research has shown that as follows to the functional examination result of the bacterial strain of the present invention:In Meng Jinna solid medium (glucose
10g, (NH4)2SO40.5g, NaCl 0.3g, MgSO4·7H2O 0.3g, FeSO40.03g, MnSO4·H2O 0.03g;
CaCO35.0g, KCl 0.3g, Ca3(PO4)225.0g, lecithin 0.3g, 18~20g of agar, pH value 7.2~7.4) on cultivate one
Zhou Hou, it is 3.5mm to measure first day its colony diameter d, and Soluble phosphorus loop diameter D is 4.5mm, D/d 1.3, its bacterium colony of third day is straight
Diameter d is 4.5mm, and Soluble phosphorus loop diameter D is that 12mm, D/d 2.7, the 7th days its colony diameter d are 5mm, and Soluble phosphorus loop diameter D is
13mm, D/d 2.6.In Phos fluid nutrient medium (C6H12O610.0g, (NH4)2SO40.5g, NaCl0.3g, KCl 0.3g,
MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, MnSO4·4H2O 0.03g, Ca3(PO4)225.0g, water 1000mL, pH
7.0-7.5.) culture 4 days after, with molybdenum blue colorimetric method measure culture solution in rapid available phosphorus changes of contents, to reacting bacteria solution
Phosphorus ability, it is 50.1mg/L to measure available phosphorus contents in JP6 culture solutions.In the R2A liquid training containing L-Trp (200mg/L)
Support base (yeast powder 0.5g, tryptone 0.5g, casamino acid 0.5g, glucose 0.5g, soluble starch 0.5g,
K2HPO40.3g, MgSO4·7H2O 0.05g, Sodium Pyruvate 0.3g.) in, 50 μ L are taken after 28 DEG C, 180R/min, shaking table culture 4d
Bacteria suspension drop adds 50 μ L Salkowski color solutions (50mL35%HClO on whiteware plate4+1mL 0.5mol/L
FeCl3) whiteware plate observes after room temperature avoid light place 30min, color redden expression can producing IAA, then with light splitting
Photometry measures the OD600 values of bacteria suspension, and bacteria suspension 10000R/min is then centrifuged 10min, takes supernatant to be added isometric
Salkowski color solutions, being protected from light standing 30min makes its colour developing, measures its OD530 value, when calculating bacteria concentration OD600 values are 1,
The concentration of IAA, it is 128.9mg/mL to measure IAA contents in JP6 zymotic fluids.The cigarette strain number of blade handled through JP6 when potting 30 days
More than control, maximum blade surface area does not reach significant difference also greater than control, the cigarette strain handled through JP6 at 60 days
Plant height, stem girth, maximum blade surface area, the number of blade are above control, cigarette strain plant height that 90 days whens are handled through JP6, the number of blade with
Control reaches significant difference, and root, stem, leaf, the total fresh weight of plant of the tobacco plant handled through JP6 are better than control treatment, and
Reach significant difference, after harvesting in the statistics of biomass it can be seen that plant root, stem, leaf dry weight and plant gross dry weight
It is better than control, while stem weight, plant gross dry weight are reaching significant difference.Compared with the control, root dry weight amplification is
60.8%, stem weight amplification is 54.5%, and leaf gross dry weight amplification is 10.1%, and plant weights amplification is 24.9%.The above research
The result shows that JP6 bacterial strains can be very good to promote tobacco growing, had potential application in terms of promoting tobacco growing.
Enterobacter cloacae (Enterobacter cloacae) JP6 of the present invention.Depositary institution:Chinese microorganism strain preservation
Administration committee's common micro-organisms center (CGMCC), address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date:
On August 28th, 2014, deposit number:CGMCC No.9615.
Description of the drawings
Fig. 1 is the cigarette strain plant height column diagram for handling for 60 days and 90 days JP6 with compareing;
Fig. 2 is the cigarette strain stem girth column diagram for handling for 60 days and 90 days JP6 with compareing;
Fig. 3 is that JP6 processing in 30 days, 60 days and 90 days accumulates column diagram with the cigarette strain maximum leaf surface compareed;
Fig. 4 is the cigarette strain number of blade column diagram that 30 days, 60 days and 90 days JP6 are handled and compareed;
Fig. 5 is that JP6 is handled and the fresh biomass column diagram of the cigarette strain compareed;
Fig. 6 is the cigarette strain dry biomass column diagram that JP6 is handled and compareed;
Fig. 7 is JP6 phosphorus decomposing design sketch.
Specific implementation mode
With reference to embodiment, the present invention is further illustrated.
Enterobacter cloacae (Enterobacter cloacae) JP6 of the present invention, depositary institution:Chinese microorganism strain is protected
Hide administration committee's common micro-organisms center (CGMCC), address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date:
On August 28th, 2014, deposit number:CGMCC No.9615.
(1) screening of CGMCC No.9615
In the process of beef extract-peptone fluid nutrient medium enrichment:Dilution plate cultivation detaches, and weighs and picks up from Hunan mulberry
Land owned by officials level ground vega fresh soil 10g is planted, is placed in the 250mL triangular flasks for including bead and 90mL sterile waters, 170r/min shakes
30min is swung, then presses 10-5、10-6、10-7Gradient is diluted, and 0.1mL is respectively taken to be coated on straw medium (fresh straw straw
121 DEG C of high pressure steam sterilization 25min of stalk 300g, agar 25g, water 1000mL.) tablet and Phos culture medium (glucose
10.0g, (NH4)2SO40.5g, NaCl 0.3g, KCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g,
MnSO4·4H2O0.03g, Ca3(PO4)225.0g, water 1000mL pH 7.0-7.5, agar 25g.) on tablet, wherein stalk is trained
It supports base and is coated with two parts, a copy of it covers one layer of water agar as thick as possible.Each gradient is repeated 3 times.Water agar will not be stamped
Straw medium tablet be positioned over 15 DEG C of culture 5-8d, the tablet and Phos culture medium flat plate for being stamped water agar be placed in 37 DEG C
Cultivate 24-48h.
By the bacterium colony percutaneous puncture-inoculation grown on water agar-straw medium in the test tube for filling semisolid straw medium
In, then above test tube one layer of about 1cm thickness of capping water agar, the atoleine of Reperfu- sion about 2cm thickness seals, manufacture entirely without
Oxygen environment.
Bacterium colony on the bacterium colony cultivated on straw medium under cryogenic conditions and Phos culture medium is inoculated into phase again
It answers and is cultivated under the same conditions on culture medium.
Above each processing is passed on 3 times, and the fast bacterium isolate purification storage of the speed of growth is chosen.Wherein phosphorus decomposing culture medium
On select the bacterium isolate containing phosphorus decomposing circle.
The bacterium isolate that secondary screening obtains is purified using three zoning collimation methods, after purification to the different shape of gained
Bacterium colony is verified by the condition in secondary screening again, and records colonial morphology.
By tablet Soluble phosphorus circle method, Soluble phosphorus circle method and molybdenum blue colorimetric method, ultraviolet spectrophotometry (survey growth cellulose content), protect
Green method, ability of dissolving potassium measuring method measure its phosphate solubilization respectively, produce auxin ability, produce ability and the potassium decomposing of the basic element of cell division
Ability.Filtered out from isolated bacterial strain one plant can phosphorus decomposing produce the bacterial strain of auxin again, be denoted as JP6.
(2) CGMCC No.9615 strain idenfications
The extraction of genomic DNA:It will be cultivated in 37 DEG C in isolated bacterial strain streak inoculation to LB solid mediums
After for 24 hours, picking single bacterium falls in the test tube equipped with LB liquid medium 37 DEG C, 150r/min shaking table cultures 12h.Specific method is joined
Read document (Ausubel FM, Brent R, Kingston RE, et al.Short Protocols in Molecular
Biology.Chichester:John Wiley&Sons,Inc,1995:36-40.).The DNA of acquirement is dissolved in 50 μ L TE to delay
In fliud flushing (10mM Tris-HCl, 1mM EDTA, PH=8.0), it is spare to be then stored in -20 DEG C of refrigerators.
16S rDNA sequence amplifications primers are synthesized by Shanghai bioengineering Co., Ltd.
Pcr amplification reaction system:Taq archaeal dna polymerases 0.5 μ L, 10 × Buffer 5 μ L, MgCl23 μ L, dNTPs
(10mmol/L) 1 μ L, primer 2 7F 1 μ L, 1 μ L of primer 1492R, template 0.5 μ L, ddH2O complements to 50 μ L.Reaction condition:95
DEG C pre-degeneration 5min;94 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of extension 1.5min, 30 recycle;72 DEG C of extension 10min.
The examining order of 16S rDNA is completed by Shanghai bioengineering Co., Ltd.
By the sequence alignment in surveyed 16S rDNA sequences and GenBank databases, the results showed that:The bacterial strain position of the present invention
In phylogenetic tree in enterobacteria (Enterobacter.sp) branch, there is nearest parent with Enterobacter cloacae
Edge relationship, 16S rRNA gene order similitudes are 99%.
The bacterial strain is in LB culture mediums (yeast extract 5g, peptone 10g, sodium chloride 10g, distilled water 1000mL, agar 15-
20g, 121 DEG C sterilizing 20min, pH7.0,37 DEG C) on culture 48h after bacterium colony it is rounded, opaque, dry tack free is light without protuberance
Yellow, neat in edge.Thalline is in spherical, does not produce gemma, Gram-negative.The physiological and biochemical property of the bacterial strain is:Methyl
Red negative, v-p negatives, Starch Hydrolysis experiment is positive, nitrate reduction test is positive, indole test is positive, vulcanization
Hydrogen experiment is positive, citrate is sent out using the experiment positive, glucose fermentation negative, sucrose fermentation test feminine gender, maltose
Ferment negative, the lactose-fermentation test positive, mannose ferment negative, glucose carbon source positive, the lactose carbon using experiment
Using experiment, positive, mannitol utilization of carbon source tests the positive, sucrose utilization of carbon source is tested positive, maltose utilization of carbon source and tested in source
The positive, citric acid utilization of carbon source negative, 2 ﹪ NaCl Salt tolerances are positive, 5 ﹪ NaCl Salt tolerances are positive, 7 ﹪ NaCl are resistance to
Salt test is positive, 10 ﹪ NaCl Salt tolerances are negative, production ammonia test is positive.By the above bacterium colony, thalli morphology, physiological and biochemical property
With 16S rDNA sequence analyses, identify that CGMCC 9615 is enterobacter cloacae (Enterobacter cloacae).
(3) phosphorus decomposing effect measuring
The inoculation that present invention screening is obtained is in Meng Jinna solid mediums (glucose 10g, (NH4)2SO40.5g,
NaCl0.3g, MgSO4·7H2O 0.3g, FeSO40.03g, MnSO4·H2O 0.03g;CaCO35.0g, KCl 0.3g, Ca3
(PO4)225.0g, lecithin 0.3g, 18~20g of agar, pH value 7.2~7.4) on cultivate one week, measure its colony diameter d, molten
Phosphorus loop diameter D calculates D/d.Phosphorus decomposing circle effect is as shown in fig. 7, it is 3.5mm, Soluble phosphorus loop diameter to measure first day its colony diameter d
D is 4.5mm, and D/d 1.3, third day its colony diameter d are 4.5mm, and Soluble phosphorus loop diameter D is 12mm, D/d 2.7, the 7th day
Its colony diameter d is 5mm, and Soluble phosphorus loop diameter D is 13mm, D/d 2.6.By JP6 in Phos fluid nutrient medium (glucose
10.0g, (NH4)2SO40.5g, NaCl 0.3g, KCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g,
MnSO4·4H2O0.03g, Ca3(PO4)225.0g, water 1000mL, pH 7.0-7.5.) in culture 4 days after, molybdenum blue colorimetric method measure
The changes of contents of rapid available phosphorus in culture solution, to the dissolving P capacity of reacting bacteria.Measuring available phosphorus contents in JP6 culture solutions is
50.1mg/L。
(4) production auxin ability measures
JP6 is connected in the R2A fluid nutrient mediums containing L-Trp (200mg/L), 28 DEG C, 180R/min, shaking table training
Support 4d.It takes 50 μ L bacteria suspensions drop on whiteware plate, while adding 50 μ L Salkowski color solutions (50mL 35%HClO4+
1mL 0.5mol/L FeCl3).The color solution of 50 μ L 50mg/L IAA will be added as positive control.Whiteware plate is in room
It is observed after warm avoid light place 30min, color reddens expression being capable of producing IAA.
The bacterium of the producing IAA of primary dcreening operation acquisition is quantitative determined, condition of culture is same as above.It is surveyed first with spectrophotometry
Determine the OD600 values of bacteria suspension, bacteria suspension 10000R/min is then centrifuged into 10min, takes supernatant that isometric Salkowski is added
Color solution, being protected from light standing 30min makes its colour developing, measures its OD530 value.When calculating bacteria concentration OD600 values are 1, the concentration of IAA.
It is 128.9mg/mL to measure IAA contents in JP6 zymotic fluids.
(5) pot experiment
It is nursery first, the tobacco seed of same kind is selected to carry out nursery.Selection is healthy and strong, disease-free, long after nursery 30 days
The consistent tobacco seedlings of gesture are transplanted, and per 1 plant of potting cigarette, mainly tobacco seedlings cane are wholly embedded into soil, pours equivalent after transplanting immediately
Water.
Followed by handled, one group of blank control, one group adds JP6 microbial inoculums.Cultivate bacteria suspension of the present invention (bacteria suspension
Bacteria concentration cfu >=108A/mL), it is used as liquid microbial inoculum, pouring root after slow seedling, every plant of tobacco seedlings add microbial inoculum 1mL.
Put basin:For influence of the difference to experimental result for reducing because of greenhouse different directions temperature, basin should hang down along with greenhouse
Histogram is to putting.
Soil moisture is paid attention in the tobacco growing phase, it is maximum to soil less than moisturizing is then needed after soil relative water content 50%
(moisturizing can be depending on the growth of cigarette strain and water shortage situation, but must assure that the irrigation amount and soil of every basin for the 80% of water-holding capacity
Humidity is consistent).
After transplanting the number of blade, plant height, stem girth, leaf color, maximum leaf length and width, growing way, residing breeding time were investigated every 10 days
Deng.When growing way appearance has notable difference between discovery processing in potting process, it will compare and handle and take pictures together with putting, simultaneously will
The root system of sampling cigarette strain carries out comparison and takes pictures.
As a result as shown in table 1, table 2, table 3, table 4, table 5
Influences of the 1. 30 days JP6 of table to tobacco agronomy character
Note:Data bit average ± standard deviation in table.The different lowercase letter indication difference conspicuousness (p with after column data<
0.05), similarly hereinafter.
Influences of the 2. 60 days JP6 of table to tobacco agronomy character
Influences of the 3. 90 days JP6 of table to tobacco agronomy character
Influences of 4. JP6 of table to cigarette strain fresh weight
Influences of 5. JP6 of table to cigarette strain dry weight
Although above-mentioned be in conjunction with the accompanying drawings and embodiments described the specific implementation mode of the present invention, not to this hair
The limitation of bright protection domain, those skilled in the art should understand that, based on the technical solutions of the present invention, this field skill
Art personnel need not make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (1)
1. an Enterobacter cloacae(Enterobacter cloacae)JP6, the bacterial strain deposit number:CGMCC No.9615.
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CN106085896B (en) * | 2016-05-10 | 2019-07-09 | 清华大学深圳研究生院 | Enterobacter cloacae and application thereof |
CN105925503B (en) * | 2016-05-17 | 2019-05-28 | 山东省林业科学研究院 | One plant of salt tolerant rhizosphere growth-promoting enterobacter cloacae and its application |
CN106318889B (en) * | 2016-08-31 | 2019-10-08 | 云南磷化集团有限公司 | A kind of Enterobacter bacterial strain and its application with dissolving P capacity |
CN106479916B (en) * | 2016-09-29 | 2020-01-03 | 普洱学院 | Enterobacter cloacae strain and application thereof |
CN108277185B (en) * | 2018-04-09 | 2020-06-23 | 中国林业科学研究院亚热带林业研究所 | Turpentine degradation microbial agent |
CN108795797B (en) * | 2018-05-24 | 2021-09-07 | 珠海市现代农业发展中心(珠海市金湾区台湾农民创业园管理委员会、珠海市农渔业科研与推广中心) | Corn root system endophytic enterobacter cloacae and application thereof |
CN109536410B (en) * | 2018-12-17 | 2020-07-17 | 石河子大学 | Salt-tolerant growth-promoting composite microbial inoculum and preparation method and application thereof |
CN111808783B (en) * | 2020-08-03 | 2022-03-04 | 山东佐田氏生物科技有限公司 | Enterobacter cloacae and production method and application of viable bacteria preparation thereof |
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