CN100451105C - Bacillus laterosporus and soil inoculation agent prepared from the strain - Google Patents
Bacillus laterosporus and soil inoculation agent prepared from the strain Download PDFInfo
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Abstract
The invention relates to Bacillus laterosporus and soil inoculation agent prepared from the stains, the conservation number of the strain is CGMCC No.1755. The soil inoculation agent is obtained by deep fermenting Bacillus laterosporus WY9701. The soil can prevent the multiplication of harmful bacteria, improve crop nutritional conditions, prevent diseases, promote plant growth and increase the production. The invention has the advantages of: 1. easy obtain of cheap raw materials, stable fermentation technique, high active viable count in fermentation product, suit for large scale industrial production, and production equipments with general purpose with other microbial bacterial agent, 2. high antireversion force product, good activation ability, and rapid propagation, and 3. good stability, long expiration life (no expire after 2-year conservation at normal temperature), simple operation, harmless to crops, no special abnormal flavor, no chafe. Bacillus laterosporus can activate and propagate rapidly at proper temperature, water and nutritional conditions to form ascendant beneficial flora and produce a marked effect.
Description
Technical field
The present invention relates to bacillus laterosporus and by the soil inoculant of this strain preparation.
Background technology
China is a large agricultural country, and agricultural is the basis of national economy, is " peace industry all over the world ".China and the world most of agricultural country is the same, all is faced with that environmental pollution, the soil acidification that fertilizer and pesticide causes hardens, soil fertility fails, agricultural product quality descends because of using in a large number at present, toxic chemical substance residually exceeds standard, the problem of sternness day by day such as harm humans health.Especially the harm that brings to the mankind of food safety, pollution of dining table has caused international community's common concern.Therefore, develop eco-agriculture, produce green food, ensure people ' s health, extremely national governments pay attention to and support." green, environmental protection, health " has become the theme of current social development.
Summary of the invention
Main purpose of the present invention provides a kind of novel bacterial of bacillus laterosporus class and by the soil inoculant of this strain preparation.
To achieve these goals, the present invention adopts following technical scheme:
To strain separated from soil identify and name for bacillus laterosporus WY9701 ((Bacilluslaterosporus WY9701). → utilize bacillus laterosporus WY9701 bacterial strain to carry out fermentating formula and fermentating condition test → carry out industrialized producing technology condition test → determine production technique → toxicity test → land for growing field crops result of use test.
Bacillus laterosporus WY9701 of the present invention on July 13rd, 2006 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCC No.1755, CGMCC No.1755 bacterium is by the bacillus laterosporus of natural separation, obtains behind strain improvement.
CGMCC No.1755 bacterial strain has following microbiological property:
1, morphological characteristic
Shape and arrangement: thalline is shaft-like, hold thin slightly, single generate to, in heaps; The gemma ovalize, (1～1.2) * (2.5～3) μ m, being attached to gemma one side has the paddle body, and gemma is adnation, middle life in the fusiform sporangiocyst.
Motion and flagellum: can move
2, physiological property
(1) gelatin stab culture:
Liquefaction: the positive; Liquefying speed: slow, a little less than
(2) starch hydrolysis: feminine gender;
(3) with the relation of oxygen: aerobic, anaerobic are all grown
(4) casein hydrolysis: the positive;
(5) tyrosine decomposes: feminine gender;
(6) formation of Protosol: feminine gender;
(7) grow among the NaCl:
2%NaCl: growth; 5,7,10%NaCl: do not grow;
Grow in (8) 0.001% N,O-Diacetylmuramidases: feminine gender
CGMCC No.1755 bacterial strain can utilize glucose, does not utilize wood sugar, pectinose
(9) nitrate reduction: the positive
3, cultivate characteristic
| Substratum | Culture condition | Cultivate and learn characteristic |
| Bacterium colony on the agar culture dish | Cultivated 1-3 days for 37 ℃ | Shape: circle: surface: lubricious, gauffer, flat or slightly the protuberance; Edge: neat or irregular slightly; Size: diameter 2～5mm; Color: white or light khaki color; Optical signature: dark, opaque |
| The agar inclined-plane: | Cultivated 1-3 days for 37 ℃ | Growth form: streak culture, there is pimple at the edge; Increment: in; Produce color: do not have; Colony colour: light khaki color; Smell: do not have obvious peculiar smell; Viscosity: not sticking |
| J-gravy | Cultivated 1-3 days for 37 ℃ | Opacity: in; Smell: free from extraneous odour; Thing sinks: not sticking, amount is few |
| The gelatin stab culture | Cultivated 8 days for 37 ℃ | Whether liquefy :+liquefying speed: slow, a little less than |
1 with the relation of temperature:
Substratum: J-gravy PH:7.3-7.5
Top temperature: 47 ℃ of optimum temperuture: 30-40 ℃ tolerable temperatures: 80 ℃ 10 minutes
Subordinate list 1, WY9701 strain characteristics
Annotate: " v " be the character instability in a bacterial strain, "+" positive reaction, and "-" negative reaction, "+/-" for a little less than the reaction, " d " is different for reaction, 11～89% the bacterial strain positive.Utilize bacillus laterosporus WY9701 to produce the preparation technology of soil inoculant
Slant strains → shake-flask seed → ferment tank → fermented liquid → super filtration → filtered liquid → adsorption dry → be mixed → finished product;
Above-mentioned culture medium prescription and culture condition:
(1) slant strains prescription: (%)
Nutrition nutrient agar substratum PH:7.0-7.5;
Culture temperature: 32-34 ℃; Time: 48-60hr
(2) shake a bottle bacterial classification prescription: (%)
Bean cake powder: 0.2-0.5 wheat bran powder: 0.8-1.2 yeast extract paste: 0.1-0.3
Sodium-chlor: 0.25-0.3 sal epsom: 0.015-0.02 dipotassium hydrogen phosphate: 0.2-0.3
Manganous sulfate: 0.005
PH:7.0-7.5 shaking speed: 160-200r/min
Culture temperature: 32-34 ℃, inoculate when cultivating 24-30hr to logarithmic phase.
(3) seed culture based formulas: (%)
Bean cake powder: 0.3-0.6 wheat bran powder: 0.8-1.5 ammonium nitrate: 0.15-0.2
Yeast extract paste: 0.2-0.4 sodium-chlor: 0.25-0.3 sal epsom: 0.015-0.02
Dipotassium hydrogen phosphate: 0.2-0.4 manganous sulfate: 0.005
PH:7.0-7.5 air flow: 1: 0.6-0.7 culture temperature: 32-34 ℃,
Culture transferring is gone into fermentor tank when being cultured to logarithmic phase.
(4) fermentative medium formula: (%)
Bean cake powder: 0.3-0.6 wheat bran powder: 0.8-1.5 ammonium nitrate: 0.15-0.2
Yeast extract paste: 0.2-0.4 sodium-chlor: 0.25-0.3 sal epsom: 0.015-0.02
Dipotassium hydrogen phosphate: 0.2-0.4 manganous sulfate: 0.005
PH:7.0-7.5 air flow: 1: 0.6-0.7
Culture temperature: 32-34 ℃, be cultured to and put jar when forming gemma more than 80%.
Super filtration among the above-mentioned preparation technology: fermented liquid is through 20 times of super filtering and concentrating, and temperature is controlled in 50 ℃, and the time is controlled in the 8hr;
Adsorption dry among the above-mentioned preparation technology: utilize light calcium carbonate, careless carbon, wood sawdust and diatomite etc. to do carrier, sorbent material: concentrated solution=(1-2): (2-3), mix after 100 ℃ of expansion dryings get the spore pulvis.
Advantage of the present invention:
1, raw material is easy to get, inexpensive, zymotechnique is stable, and leavened prod living bacteria count height is fit to large-scale industrial production, and production unit can be general with other microbiobacterial agent production units.
2, product strong stress resistance, reactivity is good, and reproduction speed is fast.Run into conditions such as suitable temperature, moisture, nutrient, just activation breeding rapidly forms the advantage profitable strain and plays a role.
3, with bacillus laterosporus WY9701 inoculation in the BCP liquid nutrient medium, 36 ± 1 ℃ of amphimicrobians are cultivated 48h, the stoste of culture and 5 times of concentrated solutions, and per os is given mouse continuous irrigation stomach 3d with 20.0ml/kgBW, observe 7d, do not observe and tried toxic reaction of mouse or death.
4, product stability is good, and validity period is long, preserves at normal temperatures not lose efficacy in 2 years, and is easy to use; Harmless to crop, no specific peculiar smell, not skin irritation.
5, through experiment and demonstration and application proof, the product applicable crops is in extensive range, and effect is remarkable.
The mechanism of action:
1., disease resistance, promote plant growth.This soil Inoculant is applied to behind the soil by the competition of space, nutrition, suppresses harmful microorganism breedings such as pathogenic bacteria; By antagonistic action, kill or suppress pathogenic bacteria, around rhizosphere, form a circle biological barrier, stop pathogenic bacteria invasion, grow surely, play the effect that reduces disease (particularly soil-borne disease).This soil Inoculant can stop the infringement of following germ virus to crop effectively:
Bacterium: Michigan clavate bacillus, carrot soft rot Erwinia, chrysanthemum Erwinia, Pseudomonas solanacearum, pseudomonas syringae, rape Zymomonas mobilis etc.; Fungi: aspergillus tubigensis,, chaetomium mould from the spore of wriggling, cephalo, thorn dish spore, sharp sickle spore, Chinese cassia tree epidemic disease are mould, citrus health phytophthora, alternaria of orange epidemic disease are mould, phytophthora parasitica, Root and stem of Cholla epidemic disease enzyme, mould, the ultimate corruption of melon and fruit corruption are mould, dry thread Pyrenomycetes, Sclerotium rolfsii, Huang wither a wheel branch spore, a Dahlia pinata chlay wheel branch spore, Verticillium etc.; Virus: tobacco mosaic virus (TMV), potato virus, cucumber mosaic virus etc.
In above-mentioned pathogenic bacterium, it is the pathogenic bacteria of the main disease of farm crop that considerable part is arranged
2., promote nutrient to decompose, increase soil fertility.This soil Inoculant is by the alternate effect, breedings such as beneficial microorganism such as phosphate solubilizing bacteria (bacillus megaterium), potassium solubilizing bacteria (silicate bacteria), yeast in the promotion soil, promote that on the one hand decomposition of soil organic matter transforms, discharge nutritive elements such as nitrogen, phosphorus, potassium, can increase the biological nitrogen fixation amount on the other hand or elements such as the phosphorus that contains in the soil mineral, potassium are decomposed, discharge, increase soil fertility, for the plant absorbing utilization, reduce the chemical fertilizer usage quantity, improve quality of agricultural product.
3., improve the soil balance fertilizing.This soil Inoculant can promote the soil beneficial microorganism to breed in a large number, keep the soil micro-ecosystem balance, the soil compaction that improvement causes because of the long-term application chemical fertilizer, acidifying situation, improve the soil physicochemical property and Soil structure, strengthen the soil water-reataining fertilizer-keeping ability, improve the level of supply of nutritive substance in the soil, make the nutrient supply equilibrium, improve utilization rate of fertilizer.
4., secrete growth substance, promotion root system development.Bacillus laterosporus is excretory various plants growth substance in its metabolic process, can promote plant root growth, increases the ability that absorbs nutrient.Its excretory nutritive substance also can directly be utilized by plant absorbing, promotes plant-growth.After handling wheat seed with the soil inoculant stoste of dilution 50 times and 100 times, its wheat seeding is compared photograph respectively and is increased 25% and 16%; Hundred strain dry weights are respectively 5.88 grams, 5.75 grams, and comparison is according to weightening finish 15.7% and 13.1% respectively.
5., the stronger bioenzyme activity of performance.Bacillus laterosporus can show higher SOD enzymic activity in metabolic process, can make the interior enzymic activity of plant materials improve 2-3 doubly.But the defensive raction of SOD enzyme involved in plant strengthens the disease-resistant resistance of plant, reduces disease, improves output.Increase mineral substance and VITAMIN such as P, K in the product, Ca simultaneously, the content of reducing sugar, amino acid etc. improves quality of agricultural product, and it is good that taste becomes, anti-storage, corrosion resistant, anti-transportation.
6., degraded soil hazardous and noxious substances, improve quality of agricultural product.This soil Inoculant has very strong Degradation to the polluted by nitrogen of soil, can make the nitrate content in the agricultural-food reduce 48.6-87.7%: this soil Inoculant also has stronger Degradation (as the residual weedicide in the degraded soil, reduce the content of chlorine in the tobacco etc.) to the hazardous and noxious substances in the soil, and the soil of deterioration played biotherapy and biological restoration effect, thereby improve the soil, quality of agricultural product is increased substantially.
Main effect and example:
(1) improve the soil:
After using soil inoculant, soil fertility, microorganism active and population structure are obviously improved.Through the soil after using is compared test shows: it can increase beneficial microorganism quantity in the soil; Reduce harmful microorganism quantity, improve the level of supply of nutritive substance in the soil.The microbe population ratio generation great variety in the soil after 30 days is used: mould, yeast+43% in same plot; Quick-acting nitrogen+13%; Available potassium+13%; Rapid available phosphorus+3%.Aerobic bacteria-42%; Total intestinal bacteria-95%; Fecal coli-99%; Positive staphylococcus-87% condenses; Salmonellas-99%; Klebsiella-99%.Use is the result prove:
Side spore sporeformer is many more in the soil, and activity is strong more, and physicochemical character is good more, and soil is fertile more.
(2) promote growth:
After farm crop were used this soil Inoculant, well developed root system, plant increased, the stem stalk increases slightly, blade increase thicken, the leaf look dark green, growth is sane.This soil Inoculant has the effect of obvious promotion cigarette strain growth.Adopting roasting preceding its plant height and leaf area all compares according to increasing.Use 20 days " Invest, Then Investigate "s of soil inoculant according to certain two places on vegetables, increase the 1.1-1.7 sheet than the number of blade of not using, plant height increases 1.8-8.6cm, and stem slightly increases 0.1-0.5cm, and just the phase of adopting is done sth. in advance 4-10 days.
(3) strengthen resistance:
1, strengthens resistance against diseases.Farm crop are used soil inoculant and all show the resistance against diseases enhancing.Be called tobacco mosaic disease, the cotton spoting verticillium wilt of " plant cancer ", obviously reduce using soil inoculant sequela rate.All show according to the test of land for growing field crops virus inoculation: use soil inoculant tobacco mosaic disease incidence and reduce 60-70%; Behind the greengrocery crop applying soil inoculant, sickness rate such as samping off, anthrax, root rot, soft rot, eqpidemic disease, Powdery Mildew, oidium, leaf spot all obviously descend, and especially club root and root knot nematode disease incidence reduce significantly.
2, strengthen cold tolerance.The somewhere suffers 30 years especially big cold waves of not meeting, and uses banana, the lichee of soil inoculant, and cold tolerance obviously strengthens, and the flap rate reaches 68%, than the raising 55% of not using.Somewhere, Zhejiang tea processing factory suffers other tea tree under the situation of serious freeze injury, uses the tea tree of soil inoculant to resist " cold spell in later spring " attack, and blade is not subjected to freeze injury, has showed stronger frost resistance ability, and output increases substantially.
3, strengthen the waterlogging-resistant ability of drought resisting.After using soil inoculant, crop is drought-resistant, waterlogging stain ability all has enhancing; No matter acid soil or alkaline soil, use soil inoculant after, the plant growth advantage is all significantly better than contrast.
(4) improve quality
Can improve the soil, balance promotes that for fertile crop steadily and surely grows, and disease is had obvious preventive effect, reduces agricultural chemicals, applying quantity of chemical fertilizer, and the degraded toxic and harmful substance is residual, thereby effectively improves quality of agricultural product.The cigarette sample is carried out check and analysis, and the tobacco tissue of using soil inoculant is loose, and first-class cigarette ratio improves 10.2%; Fragrance increases, and the taste improvement reaches conspicuous level, can improve sensory evaluating smoking's quality of tobacco leaf, helps the nuisanceless tobacco leaf of production safety.
(5) improve output:
Soil inoculant is used on different regions, Different Crop, all shows good high yield characteristic.
Crop yield obviously improves: vegetables volume increase 12-30%; Fruit volume increase about 20%; Increasing production of rice 10-15%; Tobacco volume increase 7.5-19.8%; The bright output of tealeaves increases income 26.8%, and silkworm and mulberry output increases by 20.8%.
(6) increase benefit:
Soil inoculant all kinds of farm crop are all had improve output to some extent, improve quality, listing ahead of time, reduce agricultural chemicals, fertilizer amount, reduce effects such as agricultural inputs, economic benefit is very considerable.According to statistics, the increment of greengrocery crop mu is generally 120-400 unit, and input-output ratio is 1: 12-20; The increment of cash crop mus such as tobacco is 100-540 unit, and input-output ratio is 1: 10-27; The increment of food crop mu is 50-200 unit, and input-output ratio is 1: 7-10.Tobacco is used soil inoculant, can make first-class cigarette ratio improve 5-10 percentage point, and average price improves 0.25-1.93 unit, and per mu yield can increase 72-430 unit, and input-output ratio is 1: 9-50.
Utilize the soil inoculant of bacillus laterosporus production can obviously suppress the growth and breeding of harmful germ virus, strengthen the crop root vigor, promote plant growth, to reduce the fertilizer and pesticide consumption, the protection environment has obvious effect.
Embodiment
The seed selection of embodiment 1:CGMCC No.1755 bacterium
The bacillus laterosporus wild strain of natural separation is obtained the bigger pure culture bacterial strain of a strain biomass by natural seed selection, with this bacterial strain is starting strain, its fermented liquid sporeformer number is 100,000,000/ml, use ultraviolet mutagenesis 15-50s again, obtained mutagenesis effect preferably, obtain a strain CGMCC No.1755, its fermented liquid sporeformer number is 10 times of starting strain up to 1,000,000,000/ml.
Embodiment 2, utilize CGMCC No.1755 bacterium to produce soil inoculant preparation technology to be:
CGMCC No.1755 placed shake bottle and carry out seed culture, condition is the PH:7.0-7.5 shaking speed: the 160/min culture temperature: 33 ± 1 ℃, inoculate into seeding tank when cultivating 24-30hr to logarithmic phase, in seeding tank, cultivate 24-30hr, be cultured in the fermentor tank of transferring again and form gemma more than 80%; Jar liquid obtains high density through super filtration, carrier absorption, drying process hypnocyst pulvis is put in fermentation;
Shake a bottle bacterial classification prescription (%):
Bean cake powder: 0.5 wheat bran powder: 1.2 yeast extract pastes: 0.1 sodium-chlor: 0.3
Sal epsom: 0.02 dipotassium hydrogen phosphate: 0.3 manganous sulfate: 0.005
PH:7.0-7.5 shaking speed: 160r/min culture temperature: 33 ± 1 ℃
Inoculate when cultivating 18-36hr to logarithmic phase.
Seed culture prescription (%):
Bean cake powder: 0.6 wheat bran powder: 1.5 ammonium nitrate: 0.15 yeast extract paste: 0.2 sodium-chlor: 0.25
Sal epsom: 0.015 dipotassium hydrogen phosphate: 0.4 manganous sulfate: 0.005
PH:7.0-7.5 air flow: 1: 0.6 culture temperature: 33 ± 1 ℃
Inoculate when cultivating 24-30hr to logarithmic phase.
Fermentative medium formula (%):
Bean cake powder: 0.6 wheat bran powder: 1.5 ammonium nitrate: 0.2 yeast extract paste: 0.3 sodium-chlor: 0.25
Sal epsom: 0.015 dipotassium hydrogen phosphate: 0.4 manganous sulfate: 0.005
PH:7.0-7.5 air flow: 1: 0.6 culture temperature: 33 ± 1 ℃
Be cultured to and put jar when forming gemma more than 80%.
2 aftertreatment technologys:
(1) super filtration: fermented liquid is through 20 times of super filtering and concentrating, and temperature is controlled in 50 ℃, and the time is controlled in the 8hr;
(2) adsorption dry: utilize light calcium carbonate, careless carbon, wood sawdust and diatomite etc. to do carrier adsorption ultrafiltrate liquid, sorbent material: concentrated solution=(1-2): (2-3), mix after 100 ℃ of expansion dryings get the spore pulvis.
Claims (4)
1, bacillus laterosporus WY9701, according to Zhang Jingzhong " microbial taxonomy ", according to " Bai Jie system systematic bacteriology handbook " and " bacillus ", with bacillus laterosporus ATCC64 operation mode bacterial strain, the WY9701 bacterial strain is accredited as bacillus laterosporus (Bacillus laterosporus), name for bacillus laterosporus WY9701 (Bacillus laterosporus WY9701), preserving number is CGMCCNo.1755.
2, a kind of soil inoculant is characterized in that: utilize bacillus laterosporus WY9701 bacterial strain to obtain through the microbial liquid submerged fermentation.
3, the preparation method of soil inoculant according to claim 2, it is characterized in that: CGMCCNo.1755 is placed shake bottle and carry out seed culture, condition is pH:7.0-7.5, shaking speed: 160-200r/min, culture temperature: 32-34 ℃, inoculate into seeding tank when cultivating 24-30hr, in seeding tank, cultivate 24-30hr, be cultured in the fermentor tank of transferring again and form gemma more than 80% to logarithmic phase; Fermented liquid is obtained the hypnocyst pulvis of high density through super filtering and concentrating, absorption, drying process; Wherein, described super being filtered into: fermented liquid concentrates 20 times through super strainer, and temperature is controlled in 50 ℃, and the time is controlled in the 8hr; Described adsorption dry is: do adsorbing agent carrier with light calcium carbonate, the peat composed of rotten mosses, wood sawdust and diatomite, sorbent material: concentrated solution=1～2: 2～3, mix after 100 ℃ of expansion dryings obtain the hypnocyst pulvis.
4, the preparation method of soil inoculant according to claim 3 is characterized in that: the application process of heavy wall hypnospore pulvis has: 1. admix base fertilizer or topdress in use; 2. mix in seed and use; 3. adding water waters in crop root and uses.
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| CN101228920B (en) * | 2007-07-20 | 2010-12-01 | 东莞市保得生物工程有限公司 | Application of microecologics prepared from bacillus laterosporus WY9701 on feed additive |
| CN103725626B (en) * | 2010-01-26 | 2016-04-27 | 东莞市保得生物工程有限公司 | Bacillus laterosporus preparation |
| CN102079675B (en) * | 2010-12-03 | 2013-07-17 | 董彬 | Magnetic biofertilizer and preparation method |
| CN102399710A (en) * | 2011-09-05 | 2012-04-04 | 麻林涛 | Brevibacillus laterosporus, microorganism strain agent and microorganism fertilizer prepared by using brevibacillus laterosporus as well as preparation methods thereof |
| CN102424626A (en) * | 2011-09-05 | 2012-04-25 | 麻林涛 | Microbial fertilizer prepared by bacillus laterosporus and preparation method thereof |
| CN103031260B (en) * | 2012-11-02 | 2015-07-01 | 中国农业科学院烟草研究所 | Klebsiella variicola with potassium release function, and culture method and application thereof |
| CN103289937B (en) * | 2013-06-18 | 2015-01-28 | 东莞市保得生物工程有限公司 | Method for producing bacillus laterosporus live bacteria by high density solid fermentation |
| CN106754476A (en) * | 2016-11-23 | 2017-05-31 | 北京平安福生物技术研究所有限公司 | Water-soluble vitamins microbial inoculum and its preparation method and application |
| CN106747804A (en) * | 2016-12-15 | 2017-05-31 | 中国热带农业科学院环境与植物保护研究所 | A kind of Methylotrophic gemma bacillus agent and preparation method thereof |
| CN111172064B (en) * | 2020-01-06 | 2023-01-24 | 河南新仰韶生物科技有限公司 | Bacillus laterosporus and application thereof |
| CN111733109A (en) * | 2020-07-15 | 2020-10-02 | 杨凌绿都生物科技有限公司 | Preparation and application of bacillus laterosporus microbial inoculum with growth promoting function |
| CN113186126A (en) * | 2021-04-16 | 2021-07-30 | 天津益农生物科技有限公司 | Microbial agent |
| CN113461460A (en) * | 2021-08-02 | 2021-10-01 | 黑龙江神飞生物科技股份有限公司 | Special compound fertilizer for potato crops |
| CN116000082A (en) * | 2021-10-21 | 2023-04-25 | 中国石油化工股份有限公司 | Soil remediation method based on composite microbial preparation |
| CN115074283B (en) * | 2022-06-29 | 2023-07-25 | 河南科技大学 | Brevibacillus laterosporus and application thereof |
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