CN103087925A - Application of stenotrophomonas rhizophila and cyclic dipeptide metabolite thereof - Google Patents
Application of stenotrophomonas rhizophila and cyclic dipeptide metabolite thereof Download PDFInfo
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Abstract
The invention relates to application of stenotrophomonas rhizophila and cyclic dipeptide metabolite thereof and belongs to the technical field of microbial pesticide. The stenotrophomonas rhizophila WQS37 strain is collected in the common microbial strain preservation administration center in Chinese microbial strain preservation administration committee on 5 November in 2012, and the collection number is CGMCC No.6777. The stenotrophomonas rhizophila is cultured and separated to obtain the cyclic dipeptide metabolite; and moreover, the stenotrophomonas rhizophila and cyclic dipeptide metabolite thereof and arthrobotrys oligospora are mixed, and the arthrobotrys oligospora is induced to rapidly form lots of capturing devices. The stenotrophomonas rhizophila and cyclic dipeptide metabolite can rapidly induce the arthrobotrys oligospora to form lots of capturing devices in soil, nematode can be effectively captured, and a novel thought is provided for developing a fungal microbial agent for capturing the nematode.
Description
Technical field:
The present invention relates to a kind of application of having a liking for root oligotrophic Zymomonas mobilis and ring dipeptides metabolite thereof, belong to the microbial pesticide technical field.
Background technology:
Plant nematode is one of important disease of plant, and the whole world reaches 78,000,000,000 dollars (McCarter, 2008) because of the caused farm crop financial loss of plant nematode disease every year.In China, nearly all crops such as plant nematode harm tobacco, flowers, vegetables, cotton, soybean, peanut, Chinese medicinal materials become one of limiting factor important in agriculture production.At present to mainly relying on chemical nematocides the control of nematode, but the life-time service chemical pesticide except cause environmental pollution, residual, destroy the drawback such as the eubiosis, aggravated simultaneously the resistance of insect.Utilize nemic natural enemy carry out biological control have free from environmental pollution, lasting medicine, the insect advantage such as be difficult for developing immunity to drugs has obtained the attention of national governments.
Nematode-trapping fungi as the Important Natural Enemy of pathogenic nematode, is the important biomolecule factor of occurring in nature control, balance and adjusting Nematodes popula tion, is also main source (Siddiqui and Mahmood, 1996 of research and development biological mematocide; Nordbring-Hertz et al., 2000).Approximately there be more than 200 kind of fungi to utilize trapping organs to come nematode-trapping in soil.By in-plant contact nematode, hypha,hyphae can digest nematode after after several hours, the formation trapping organs sticks, penetrates, kills.The organ of these differentiation comprises three-dimensional bacterium net, viscosity branch, slimeball and shrunk ring etc.Therefore form trapping organs extremely important concerning these fungies, thereby entered the parasitic stage.Formation for trapping organs, what people mainly paid close attention to is inducing of nematode, the substance that is called as " Nemin " that separates from the nutrient solution of living nematode and dead wire worm is induced, perhaps under laboratory condition, when nutritive deficiency, some little peptides or amino acid also can be induced, and the pheromone dormin of plant also can be induced, yet the activity of these materials is all less than living nematode high (Xu et al., 2011).In soil, the nematode-trapping fungi competitive power is weak and affected by environment larger, growth is restricted, even perhaps reached some amount, owing to can not effectively forming trapping organs, cause the nematode-trapping fungi can not nematode-trapping before the nematode infection plant root, reduced preventive effect (Jaffee, 2003).Therefore studying Nematophagous fungi quick its preventive effect of factor pair raising that forms trapping organs in soil is an important approach.
Bibliographical information was just arranged as far back as 1962, and when nematode-trapping fungi being positioned in mycostasis soil, Nematophagous fungi can directly form trapping organs from spore, studies show that in recent years, and bacterium is the key factor that affects mycostasis soil.The extract of rhizosphere soil and soil also is in the news and can induces Nematophagous fungi to form trapping organs.Although these studies show that the bacterium in soil is relevant to Nematophagous fungi formation trapping organs, these authors think that the formation of trapping organs is the result (Persmark and Nordbring-Hertz, 1997) of competition nutrition between microorganism.Up to the present only have Li Lei (Liet al., 2011) report Chryseobacterium sp.TFB to form trapping organs by inducing nematode-trapping fungi.Zygosaccharomyces (Stenotrophomonas) extensively exists in the rhizosphere of a lot of plants usually in soil, and is considered to a kind of potential biocontrol microorganisms of nematodiasiss that is used for preventing and treating.And have a liking for root oligotrophic Zymomonas mobilis (Stenotrophomonas rhizophila) owing to not finding to make human infection's disease be considered to the potential biocontrol microorganisms of a kind of tool (Hayward et al., 2009).We study root oligotrophic Zymomonas mobilis (Stenotrophomonas rhizophila) can be induced Nematophagous fungi under the effect of its meta-bolites the type species of having a liking for of finding in soil--and few spore Arthrobotrys and multiple Nematophagous fungi form a large amount of trapping organs fast, this type of research has no the open source literature report.
Reference:
1)McCarter?JP(2008)Nematology:terra?incognita?no?more.Nature?Biotechnology26:882-884
2)Siddiqui?ZA,Mahmood?I(1996)Biological?control?of?plant?parasitic?nematodes?by?fungi:a?review.Bioresource?Technology58:229-239.
3)Nordbring-Hertz?B,Jansson?HB,Tunlid?A(2000)Nematophagous?fungi.In:Encyclopedia?of?Life?Science.Macmillam?Publishers?Ltd.,Basingtoke.
4)Xu?LL,Lai?YP,Wang?L,Liu?XZ(2011)Effects?ofabscisic?acid?and?nitric?oxide?on?trap?formation?and?trapping?ofnematodes?by?the?fungus?Drechslerella?stenobrocha?AS6.1.Fungal?Biology115:97-101
5)Jaffee?BA(2003)Correlations?between?most?probable?number?and?activity?of?nematode-trapping?fungi.Phytopathology93:1599-1605.
6)Persmark?L,Nordbring-Hertz?B(1997)Cornidial?trap?formation?of?nematode-trapping?fungi?in?soil?and?soil?extracts.FEMS?Microbiology?Ecology22:313-323
7)Li?L,Ma?MC,Liu?YJ,Zhou?JW,Qu?Q,Lu?KP,Fu?DG,Zhang?KQ(2011)Induction?of?trap?formation?in?nematode-trapping?fungi?by?a?bacterium.FEMS?Microbiology?Letter322:157-165
8)Hayward?A.C.,Fegan?N.,Fegan?M.,Stirling?G.R.(2009)Stenotrophomonas?and?Lysobacter:ubiquitous?plant-associated?gamma-proteobacteria?of?developing?significance?in?applied?microbiology.Journal?ofApplied?Microbiology.108:756-770
Summary of the invention:
The object of the present invention is to provide a kind of application of having a liking for root oligotrophic Zymomonas mobilis and ring dipeptides metabolite thereof, thereby they can induce few spore Arthrobotrys to form fast a large amount of trapping organs nematode-trappings, for exploitation biological pesticide preparation provides new method.
The present invention is achieved in that
Produce bacterial strain for having a liking for root oligotrophic Zymomonas mobilis (S.rhizophila WQS3), this bacterial strain was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 5th, 2012; Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica; Preserving number: CGMCCNo:6777;
To have a liking for root oligotrophic Zymomonas mobilis liquid nutrient medium normal fermentation, fermented liquid is centrifugal to be removed after thalline with the ethyl acetate extraction that contains the 0.1mL/L trifluoroacetic acid three times, ethyl acetate extract is concentrated into paste with Rotary Evaporators under 50 degree, paste substance is separated the active fractions that obtains containing four ring dipeptides with Sephadex LH-20, after active fractions separated repeatedly with SephadexLH-20, the separation of recycle silicon glue obtained four ring two peptide monomers.
Have a liking for four ring dipeptide compounds of root oligotrophic Zymomonas mobilis and identify that with nucleus magnetic resonance and high resolution mass spectrum its structure of its structure is as follows:
These four compounds are respectively (1) Cyclo (L-leucyl-trans-4-hydroxy-L-proline); (2) Cyclo (L-Ala-L-proline); (3) cyclo-(L-phenylalanyl-trans-4-hydroxy-L-proline); (4) cyclo-(L-phenylalanyl-cis-4-hydroxy-L-prolinne).
The preparation of these 4 kinds of compounds:
(1) will have a liking for root oligotrophic Zymomonas mobilis liquid nutrient medium normal fermentation, the formula of liquid nutrient medium is: peptone 5g, extractum carnis 3g, NaCl5g, distilled water 10000l, pH7.2;
(2) fermented liquid was removed thalline in centrifugal 10 minutes under 8000rpm, with fermented liquid be concentrated into original volume 1/10 after, with isopyknic ethyl acetate extraction that contains the 0.1mL/L trifluoroacetic acid three times.Get medicinal extract after ethyl acetate extract is concentrated.Medicinal extract is dissolved in methyl alcohol, with Sephadex LH-20 wash-out under methyl alcohol, connect 1 in every 15 seconds, collect liquid 1ml for every bottle, every bottle of elutriant collecting is detected (developping agent: chloroform: methyl alcohol: water 6: 2: 0.4 with the silica gel G thin-layer chromatography; The sulfuric acid colour developing), be that elutriant between 0.2-0.6 merges concentrated with the Rf value, obtain containing the active fractions of these 4 ring dipeptides, active fractions is with Sephadex LH-20 wash-out repeatedly under methyl alcohol, then use silica gel G at chloroform: separating for several times under methyl alcohol, namely obtain compound 1-4, with the structure of nucleus magnetic resonance and Mass Spectrometric Identification compound.
The present invention separates has a liking for the experiment that root oligotrophic Zymomonas mobilis and 4 ring dipeptide compounds are used for inducing few spore Arthrobotrys formation trapping organs:
(1) preparation mycelia surface band is had a liking for few spore Arthrobotrys of root oligotrophic Zymomonas mobilis (referred to as AO
Sr): will lack the spore Arthrobotrys and be inoculated in liquid CMB, and cultivate 7 days under 28 ℃, the CMB culture medium prescription is: Semen Maydis powder: 20 grams, 1000 milliliters, water, pH nature.To have a liking for root oligotrophic Zymomonas mobilis and cultivate 48 hours under 28 ℃ with liquid nutrient medium, the formula of liquid nutrient medium is: peptone 5g, extractum carnis 3g, NaCl5g, distilled water 1000ml, pH7.2.Get the fermented liquid that 0.1-2.0ml has a liking for root oligotrophic Zymomonas mobilis and put into few spore Arthrobotrys of cultivating 7 days, cultivated again after mixing 1-7 days.Then with AO
SrObtain mycelia with gauze after with filtering fermentation liquor.
(2) have a liking for root oligotrophic Zymomonas mobilis with what fluorescence in situ hybridization technique detected few spore Arthrobotrys mycelia surface: with the GCTCAT TCT TTC GCG CCA GG-3 ' of the probe pB-02285[5 ' that can detect stenotrophomonas (Stenotrophomonas)-(FITC)] bacterium that detects the surface that is present in few spore Arthrobotrys mycelia has a liking for root oligotrophic Zymomonas mobilis.We can find out from Fig. 1, at AO
SrThe mycelia surface exists has a liking for root oligotrophic Zymomonas mobilis.
(3) 4 ring two peptide monomers are dissolved in respectively in a small amount of DMSO, then are made into 100 μ g/ μ l with sterilized water, stand-by.
(4) 4 ring dipeptide compounds are induced AO
SrForm the flat board experiment of trapping organs: put into the CMB of 10 times of 3 milliliters of dilutions in the flat board of 3 centimetres of diameters, with the AO of 0.5 centimetre of inoculating needle picking diameter
SrThen mycelia gets 5 μ l compounds and puts into flat board, mixes, and observes mycelia after 14 hours, can see having formed a large amount of trapping organs on mycelia, sees Fig. 2.
(5) ring two peptide monomers and AO
SrThe determination of activity of nematode-trapping in soil after mixing: do test with 10 centimetres of flat boards, put the soil of 10 gram sterilizations in each flat board, will encircle two peptide monomers and AO
SrMycelia fully mixes, and ratio is: put 0.2-3.0 milligram ring two peptide monomers in every gram mycelia.Put into respectively few spore Arthrobotrys mycelia in flat board, AO
SrMycelia, ring two peptide monomers and AO
SrThe mycelia that mixes, blank is sterile soil.After at room temperature placing 15 hours, put into 300 nematodes, after 3 days, soil is taken out, with Büchner funnel, nematode is washed out, calculate the predation rate.
With every gram AO
SrIt is example that the ratio that mycelia mixes with ring two peptide monomers is 0.2 milligram, and what blank group nematode was dead is 7%, and the predation rate of few spore Arthrobotrys is 14%, AO
SrThe predation rate be 30%, 4 ring two peptide monomers and AO
SrThe predation rate that mycelia mixes is respectively 62%, 61%, 60%, 63%.Compound 1-4 and AO are described
SrCan effective nematode-trapping in soil after mixing.
Experiment shows: have a liking for root oligotrophic Zymomonas mobilis and 4 ring dipeptides metabolites and can induce few spore Arthrobotrys to form fast a large amount of trapping organs.
Of the present inventionly have a liking for root oligotrophic Zymomonas mobilis and its ring dipeptides metabolite and can form a large amount of trapping organs by the few spore Arthrobotrys of rapid induction in soil, effectively nematode-trapping, provide new thinking for developing the nematode-trapping fungi bacteria agent.
Description of drawings:
There is the root of having a liking for oligotrophic Zymomonas mobilis on the mycelia surface that Fig. 1 is presented at few spore Arthrobotrys.
Fig. 2 demonstration finds out that few spore Arthrobotrys has formed a large amount of mycelia and caught device.
Embodiment:
Preserving number be CGMCC No:6777 have a liking for root oligotrophic Zymomonas mobilis (S.rhizophilaWQS37), use the liquid nutrient medium normal fermentation, fermented liquid is centrifugal to be removed after thalline with the ethyl acetate extraction that contains the 0.1mL/L trifluoroacetic acid three times, ethyl acetate extract is concentrated into paste with Rotary Evaporators under 50 degree, paste substance is separated the active fractions that obtains containing four ring dipeptides with Sephadex LH-20, after active fractions separated repeatedly with SephadexLH-20, the separation of recycle silicon glue obtained four ring two peptide monomers.
Embodiment one:
The present invention is separated to from soil and has a liking for root oligotrophic Zymomonas mobilis and its ring dipeptides metabolite and be used for inducing few spore Arthrobotrys to form the experiment of trapping organs:
1, preparation AO
Sr: will lack the spore Arthrobotrys and be inoculated in liquid CMB, and cultivate 7 days under 28 ℃, the CMB culture medium prescription is: Semen Maydis powder: 20 grams, 1000 milliliters, water, pH nature.To have a liking for root oligotrophic Zymomonas mobilis and cultivate 48 hours under 28 ℃ with liquid nutrient medium, the formula of liquid nutrient medium is: peptone 5g, extractum carnis 3g, NaCl5g, distilled water 1000ml, pH7.2.Get the fermented liquid that 0.1ml or 1ml have a liking for root oligotrophic Zymomonas mobilis and put into few spore Arthrobotrys of cultivating 7 days, cultivated again after mixing 7 days or 4 days.Then with AO
SrObtain mycelia with gauze after with filtering fermentation liquor.
2, detect AO with fluorescence in situ hybridization technique
SrThe mycelia surface have a liking for root oligotrophic Zymomonas mobilis: with the GCT CAT TCT TTCGCG CCA GG-3 ' of the probe pB-02285[5 ' that can detect stenotrophomonas (Stenotrophomonas)-(FITC)] detect the surface that is present in few spore Arthrobotrys mycelia have a liking for root oligotrophic Zymomonas mobilis.Can find out from Fig. 1, at AO
SrThe mycelia surface exists has a liking for root oligotrophic Zymomonas mobilis.
3, ring two peptide monomers and AO
SrThe determination of activity of nematode-trapping in soil after mixing: do test with 10 centimetres of flat boards, put the soil of 10 gram sterilizations in each flat board, will encircle two peptide monomers and AO
SrMycelia fully mixes, and ratio is: put 0.2 milligram or 1.2 milligrams of ring two peptide monomers in every gram mycelia.Put into respectively few spore Arthrobotrys mycelia in flat board, AO
SrMycelia, ring two peptide monomers and AO
SrThe mycelia that mixes, blank is sterile soil.After at room temperature placing 15 hours, put into 300 nematodes, after 3 days, soil is taken out, with Büchner funnel, nematode is washed out, calculate the predation rate, the predation efficiency method of calculation are:
Predation rate %=dead wire borer population/300 * 100
Take sterile soil as contrast, whole experiment triplicate is got three mean value calculation and is gone out average mortality.
4, test-results
The activity of few spore Arthrobotrys sample nematode-trapping in soil of table 1 different treatment
Result shows: can induce few spore Arthrobotrys to form effectively nematode-trapping of trapping organs thereby have a liking for root oligotrophic Zymomonas mobilis and its ring dipeptides metabolite.
Embodiment two:
The present invention is separated to from soil and has a liking for root oligotrophic Zymomonas mobilis and its ring dipeptides metabolite and be used for inducing few spore Arthrobotrys to form the experiment of trapping organs:
1, preparation AO
Sr: will lack the spore Arthrobotrys and be inoculated in liquid CMB, and cultivate 7 days under 28 ℃, the CMB culture medium prescription is: Semen Maydis powder: 20 grams, 1000 milliliters, water, pH nature.To have a liking for root oligotrophic Zymomonas mobilis and cultivate 48 hours under 28 ℃ with liquid nutrient medium, the formula of liquid nutrient medium is: peptone 5g, extractum carnis 3g, NaCl5g, distilled water 1000ml, pH7.2.Get the fermented liquid that 2.0ml has a liking for root oligotrophic Zymomonas mobilis and put into few spore Arthrobotrys of cultivating 7 days, cultivated again after mixing 3 days.Then with AO
SrObtain mycelia with gauze after with filtering fermentation liquor.
2, with fluorescent in situ technology for detection AO
SrThe mycelia surface have a liking for root oligotrophic Zymomonas mobilis: with the GCT CAT TCT TTC GCGCCA GG-3 ' of the probe pB-02285[5 ' that can detect stenotrophomonas (Steno trophomonas)-(FITC)] detect and be present in AO
SrThe mycelia surface have a liking for root oligotrophic Zymomonas mobilis.Can find out from Fig. 1, at AO
SrThe mycelia surface exists has a liking for root oligotrophic Zymomonas mobilis.
3, ring two peptide monomers and AO in soil
SrThe determination of activity of nematode-trapping after mixing: do test with 10 centimetres of flat boards, put the went out soil of bacterium of 10 grams in each flat board, will encircle two peptide monomers and AO
SrMycelia fully mixes, and ratio is: put 3.0 milligrams of ring two peptide monomers in every gram mycelia.Put into respectively few spore Arthrobotrys mycelia in flat board, AO
SrMycelia, ring two peptide monomers and AO
SrThe mycelia that mixes, blank is sterile soil.After at room temperature placing 15 hours, put into 300 nematodes, after 3 days, soil is taken out, with Büchner funnel, nematode is washed out, calculate the predation rate, the predation efficiency method of calculation are:
Predation rate %=dead wire borer population/300 * 100
Take sterile soil as contrast, whole experiment triplicate is got three mean value calculation and is gone out average mortality.
4, test-results
The activity of few spore Arthrobotrys sample nematode-trapping in soil of table 2 different treatment
Result shows: can induce few spore Arthrobotrys to form effectively nematode-trapping of trapping organs thereby have a liking for root oligotrophic Zymomonas mobilis and its ring dipeptides metabolite.
Claims (1)
1. have a liking for the application of root oligotrophic Zymomonas mobilis and ring dipeptides metabolite thereof, it is characterized in that:
(1) with preserving number be CGMCC No:6777 have a liking for root oligotrophic Zymomonas mobilis (S. rhizophila WQS37), use solution culture fermentation, fermented liquid is centrifugal to be removed after thalline with the ethyl acetate extraction that contains 0.1 mL/L trifluoroacetic acid three times, ethyl acetate extract is concentrated into paste with Rotary Evaporators under 50 degree, paste substance is separated the active fractions that obtains containing four ring dipeptides with Sephadex LH-20, after active fractions separated repeatedly with Sephadex LH-20, the separation of recycle silicon glue obtained four ring two peptide monomers;
(2) preparation mycelia surface band is had a liking for few spore Arthrobotrys of root oligotrophic Zymomonas mobilis:
A. the spore Arthrobotrys is inoculated in liquid CMB less, cultivates 7 days under 28 ℃, and the CMB culture medium prescription is: Semen Maydis powder: 20 grams, 1000 milliliters, water, pH nature;
B. have a liking for root oligotrophic Zymomonas mobilis and cultivated 48 hours under 28 ℃ with liquid nutrient medium, the formula of liquid nutrient medium is: peptone 5 g, extractum carnis 3 g, NaCl 5 g, distilled water 1000ml, pH 7.2;
C. get the fermented liquid that 0.1-2.0 ml has a liking for root oligotrophic Zymomonas mobilis and put into few spore Arthrobotrys of cultivating 7 days, cultivated again after mixing 1-7 days; Then the surface is obtained with the mycelia of having a liking for root oligotrophic Zymomonas mobilis after with filtering fermentation liquor with gauze with few spore Arthrobotrys mycelia of having a liking for root oligotrophic Zymomonas mobilis;
(3) band that (2) step is obtained is had a liking for few spore Arthrobotrys mycelia of root oligotrophic Zymomonas mobilis and the ring dipeptides metabolite that (1) step obtains, be placed in soil by putting the mixing of 0.2-3.0 milligram ring dipeptides metabolite in every gram mycelia, as inducing few spore Arthrobotrys to form fast the application of a large amount of trapping organs.
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| CN106148221A (en) * | 2016-01-19 | 2016-11-23 | 沈阳农业大学 | The stenotrophomonas antibacterial of one strain induction Semen arachidis hypogaeae root-knot nematode resistant |
| CN110106117A (en) * | 2019-05-17 | 2019-08-09 | 贵州大学 | Regional efficient nitrogen microbial composite bacteria agent and its application in a kind of suitable Guizhou Province |
| CN112522115A (en) * | 2020-12-09 | 2021-03-19 | 云南大学 | Application of microbacterium Paraoxidans in inducing Arthrobotrys oligospora to generate predatory organ and method |
| CN113736701A (en) * | 2021-09-06 | 2021-12-03 | 江苏省中国科学院植物研究所 | Root-philic oligo-oxygen monad and application thereof |
| CN115747104A (en) * | 2022-11-14 | 2023-03-07 | 云南大学 | Application of brevundimonas in inducing Arthrospora oligospora to generate predatory organ |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148221A (en) * | 2016-01-19 | 2016-11-23 | 沈阳农业大学 | The stenotrophomonas antibacterial of one strain induction Semen arachidis hypogaeae root-knot nematode resistant |
| CN106148221B (en) * | 2016-01-19 | 2020-01-17 | 沈阳农业大学 | Stenotrophomonas bacteria for inducing peanuts to resist root-knot nematode disease |
| CN110106117A (en) * | 2019-05-17 | 2019-08-09 | 贵州大学 | Regional efficient nitrogen microbial composite bacteria agent and its application in a kind of suitable Guizhou Province |
| CN112522115A (en) * | 2020-12-09 | 2021-03-19 | 云南大学 | Application of microbacterium Paraoxidans in inducing Arthrobotrys oligospora to generate predatory organ and method |
| CN112522115B (en) * | 2020-12-09 | 2022-09-02 | 云南大学 | Application of microbacterium Paraoxidans in inducing Arthrobotrys oligospora to generate predatory organ and method |
| CN113736701A (en) * | 2021-09-06 | 2021-12-03 | 江苏省中国科学院植物研究所 | Root-philic oligo-oxygen monad and application thereof |
| CN115747104A (en) * | 2022-11-14 | 2023-03-07 | 云南大学 | Application of brevundimonas in inducing Arthrospora oligospora to generate predatory organ |
| CN115747104B (en) * | 2022-11-14 | 2024-04-16 | 云南大学 | Application of Brevundimonas in inducing Arthropoda shapefaciens to produce predatory organs |
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