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Effects of Biogas Slurry Application on the Soil Microorganisms of P.americanum × P.purpureum Grassland

沼液对狼尾草地土壤微生物群落的影响



全 文 :!24" !1#
 Vol.24  No.1
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犱狅犻:10.11733/j.issn.10070435.2016.01.008
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犈犳犳犲犮狋狊狅犳犅犻狅犵犪狊犛犾狌狉狉狔犃狆狆犾犻犮犪狋犻狅狀狅狀狋犺犲犛狅犻犾犕犻犮狉狅狅狉犵犪狀犻狊犿狊狅犳
犘.犪犿犲狉犻犮犪狀狌犿×犘.狆狌狉狆狌狉犲狌犿犌狉犪狊狊犾犪狀犱
ZHONGZhenmei1,2,SONGYana3,HUANGXiusheng1,2,YOUXiaofeng1,2,WENGBoqi1,2,
HUANGQinlou2,4,CHENZhongdian1,2,FENGDeqing1,2
(1.AgriculturalEcologyInstitute,FujianAcademyofAgriculturalSciences(FAAS),Fuzhou,FujianProvince350003,China;
2.FujianEngineeringandTechnologyResearchCenterforHilyPrataculture,Fuzhou,FujianProvince350003,China;
3.Instituteofbiotechnology,FAAS,Fuzhou,FujianProvince350003,China;
4.InstituteofAnimalHusbandryandVeterinaryMedicine,FAAS,Fuzhou,FujianProvince350003,China)
犃犫狊狋狉犪犮狋:Inordertorevealtheeffectsofbiogasslurryapplicationonimprovingsoilfertility,thechangeof
soilmicroorganismsunderdifferentamountsofbiogasslurry(equalto200,150,100and50kgN·ha-2
pureN)applicationon犘犲狀狀犻狊犲狋狌犿.犪犿犲狉犻犮犪狀狌犿×犘犲狀狀犻狊犲狋狌犿.狆狌狉狆狌狉犲狌犿grasslandwasstudiedbychlor
oformfumigation,fluorescencequantitativePCRandPCRDGGEmethodsbasedonfieldexperiment.The
resultsshowedthatsoilmicrobialbiomassCandNof4treatmentssignificantlyincreasedbybiogasslurry
applicationcomparedtocontrol,andtherewasasignificantcorrelationbetweentheslurryapplicationa
mountandtheincrementofsoilmicrobialbiomassCandN.Biogasslurrysignificantlyincreasedtheabun
danceof16SrDNAandSSUrDNAgenewhichwassignificantlyrelatedwiththeapplicationamountof
biogasslurry,andthequantitiesof16SrDNAcontainingbacterialandSSUrDNAcontainingfungiwere
themaximuminthetreatmentofcontaining200kgN·ha-2biogasslurry,whiletheabundanceof狀犻犳犎
containinggenesignificantlydecreased.Asaresult,itcouldconcludethattheapplicationofbiogasslurry
hasnosignificanteffectonthecommunitydiversityofsoilbacteria,fungiandnitrogenfixingbacteria,but
promotesthegrowthofmicroorganismscontaining16SrDNAgeneandSSUrDNAgenes,inhibitsbacteri
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algrowthcontaining狀犻犳犎gene,andchangesthenumberofmicroorganisms.
犓犲狔狑狅狉犱狊:Biogasslurry;Bacterial;Fungi;Azotobacter;Geneabundance;Microbialcommunitydiversity
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Table1 Thedesigntableofrandompermutationtest
BCÓùij Arrangementoftheexperimenttreatment
!ߑ Thefirstline T5 T4 T3 T2 T1
!å‘ Thesecondline T2 T5 T4 T1 T3
!ñ‘ Thethirdline T1 T3 T5 T2 T4
1.4.2 @AÀlm^_`]aŒ ÙCDNA
›f

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gene)TBÜßÆ}ÏBÜߛœTÒâ›fÙC
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—ÞSSUrDNA®56êiÞ狀犻犳犎 ®5T8ºÊ
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PCRSystemiíTS\‘^DZ‘©`PCR=•。
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ÞSSUrDNA®56êiÞ狀犻犳犎 ®5•äF]F
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_ÊKÜ1L5pmol·L-1,°_9KÃĺp,S
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68LQÚQ。
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ÞSSUrDNA ®56êiÞ狀犻犳犎 ®58ºÊ
K
[1719]
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±TAE^Ù10000±)¨ Wµýµp45min,dBio
Rad’l]^mÒ。
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Table2 PrimersandPCRconditionsusedforthestudy
µF9K
Target
group
ÊK
Primer
lj
Sequence(5′-3′)
PCRuŠÍÖ
ThermalprofileforPCRDGGE
Z‘©`PCRuŠÍÖ
ThermalprofileforrealtimePCR
ž^Š}
Reference

Bacteria
F341
F341GC
R534
CCTACGGGAGGCAGCAG
ATTACCGCGGGTGCTGG
94℃S<5min,94℃<1
min、55℃ïn2min672℃Á
o2min,35MGH,72℃10min
95℃S<30s,94℃<5s,
60℃ïnÁo34s,40MGH
Muyzer
犲狋犪犾.,
1993
—Þ
Fungi
FF390
FR1FR1GC
CGATAACGAACGAGACCT
AICCATTCAATCGGTAIT
95℃S<8min,95℃<30
s、50℃ïn45s672℃Áo2
min,35MGH,72℃10min
95℃S<30s,94℃<30
s,50℃ïn45s,72℃Áo2
min,40MGH
Vainioand
Hantul,
2000
êiÞ
N2fixer
PolF
PolFGC
AQER
TGCGAYCCSAARGCBGACTC
GACGATGTAGATYTCCTG
94℃S < 5 min,94℃ <  1
min、55℃ïn1min672℃Á
o2min,35MGH,72℃10min
95℃S<30s,94℃<5s,
60℃ïnÁo34s,40MGH
Poly
犲狋犪犾.,
2001
  ü:g˜=¾LF341,FR16PolFÊKT5′?îßMGCp
Note:indicateGCclamp(5′CCGCCGCGCGGCGGGCGGGGCGGGGGCACGGGG3′)wasattachedtothe5′endofprimersF341,
FR1andPolF
1.5 lm>r
dQuantityOne(BioRad)ÕÖDDGGEë[
&DNAÍÜTŸÂ6~A\‘ë;ð,²ß?9
KOP:»ëShannonWeaverindex(H′)=
((n/N)×ln(n/N)(n=¿MÜT~A,N=@ß
:11JÜ~AT6
)[20]。
ÏdSPSS18.0^²Õ
Ö\‘ÒØ
(ANOVA)=•6ˆ‰=•,^ ²Ò
ânLSD•¸t,غ…†QR犘<0.05。
2 pq>r
2.1 š›oQÏÓ、J,1CwN
ädAá_

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,0~20cm620~40
cmْÙCài、àj、6àk©„íîóI,IØ
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Table3 Thebasicphysicalandchemicalpropertiesofsoil
Óù
Treatment
0~20cm 20~40cm
pH
pHvalue
ài TN
/g·kg-1
JÁF
Organic
matter
/g·kg-1
àj TP
/g·kg-1
àk TK
/g·kg-1
pH
pHvalue
ài TN
/g·kg-1
JÁF
Organic
matter
/g·kg-1
àj TP
/g·kg-1
àk TK
/g·kg-1
T1 4.45±0.24a 0.67±0.08a12.82±1.96c 0.34±0.04a 55.5±0.502b 4.36±0.04a 0.81±0.25a 11.95±2.46c 0.34±0.12a 55.5±0.502b
T2 5.12±0.31a 0.73±0.08a14.15±1.26bc0.43±0.07a 55.8±0.212b 4.58±0.11a 0.62±0.03a 13.90±1.66bc0.44±0.03a 55.8±0.212b
T3 4.74±0.23a 0.89±0.15a 17.20±4.17a 0.34±0.08a 56.7±0.472a 4.43±0.08a 0.72±0.10a 14.96±1.45ab0.42±0.05a 56.7±0.472a
T4 4.55±0.30a 0.82±0.042a15.11±0.95ab0.37±0.05a 57.6±0.556a 4.48±0.08a 0.90±0.11a 18.64±3.05a 0.49±0.02a 57.6±0.556a
T5 4.75±0.31a0.84±0.13a 17.10±1.67a 0.40±0.02a 56.3±0.272a 4.52±0.05a 0.73±0.20a 15.17±2.83ab0.37±0.06a 56.3±0.272a
  ü:@j6@uU;Çg˜6@Óù%غ…†(犘<0.05),œ@
Note:Differentlowercasesinthesamecolumnindicatesignificantdifferenceatthe0.05level.Thesameasbelow
  AáïX-__ÙC?9K`C(SMBC)6N
(SMBN)~g41˜。0~20620~40cmْ
ÙC?9K`N6C©ÀìAáBï`Tíîn
íî

‘& T5W6Bï(iCÚ`n:200kg·
hm-2)ÉÃ-_ÙC?9K9K` N6CÝs。
0~20cmْ,T2~T5Óù?9K` N©…†
séDÒT1Óù,T46 T5ÓùA…†sé T2
Óù

‘XÓùã%(غ
;T46T5Óù
?9K`C…†séDÒ6‘XÓù,n T46
T5ã%غAWd…†QR。ghAáBï§
íî0~20cmْÙC?9K`。20~40cmÙ
’
,T5Óù?9K` N…†séDÒ6‘XÓ
ù

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ˆ‰=•gh

AáBïQR?9K`C6
NTˆ‰]ë=¾n0.83360.824,©Wdú…
†QR
(犘<0.01)(g5)。
`4 c~š_š›犛犕犅犖w犛犕犅犆
Table4 SMBNandSMBCofdifferentsoillayers
Óù Treatment
0~20cm 20~40cm
SMBN/mg·kg-1 SMBC/mg·kg-1 SMBN/mg·kg-1 SMBC/mg·kg-1
T1(CK) 9.02±1.81c 63.74±9.91d 7.25±0.90b 58.32±1.94b
T2 22.78±2.72b 136.40±43.81cd 8.58±1.55b 263.20±14.53a
T3 34.41±3.98ab 172.67±34.96c 15.98±7.64b 145.87±2.84a
T4 43.81±0.16a 377.59±88.44b 14.76±3.14b 117.12±1.394a
T5 38.92±12.71a 524.62±8.68a 35.28±9.86a 195.98±3.09a
2.2 š›J,Nø`€
2.2.1 @AØÙ16SrDNAڎÛI ÏdZ‘
©`PCR¾¨DHÞ16SrDNA®5ëA\‘=
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ùHÞ16SrDNA®5ëA…†séT1(DÒ)|
‘XÓù
,T36T4ÓùA…†séDÒ6T2Ó
ù
,0~5610~15cmْT26T1ã%غA
Wd…†QR

n15~20620~25cmْ T4
6T5Óù…†séT1,T26T3Óù,T26T3
T1ã%AWd…†QR。ghAáBï_§í
î0~25cmْÙCHÞ®5qëë,[\»16S
rDNA®5T?9KT…I,"#»16SrDNA®
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AáBïQ
RÙCHÞ16SrDNA®5ëATˆ‰]ën
0.968,Wdú…†QR(犘<0.01)(g5)。
=1 ab16犛狉犇犖犃Nø`€
Fig.1 Geneabundanceof16SrDNA
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Fig.2 GeneabundanceofSSUrDNA
=3 dKb狀犻犳犎 Nø`€
Fig.3 Geneabundanceof狀犻犳犎
2.2.2 @AÜÙSSUrDNAڎÛI èë2L
ã

ÀìAáBï`Tíî

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®5ëAA„íîóI
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C—ÞSSUrDNA ®5ëA…†sé T1,T26
T3Óù,0~565~10cmْT26T3ÓùA…
†séDÒT1Óù,n10~15,15~20620~25
cmْT26T3ÓùDÒT1ã%غ6…†。
ghAáBï_§íî0~25cmْÙC—Þ®
5qëë

—Þc`íî

ò.Gíî:ŠLG’
gPónh…

Àìْ‚Aíî.Gcd„€
óI

ˆ‰=•gh

AáBïQRÙC—Þ
SSUrDNA®5ëATˆ‰]ën0.972,Wdú
…†QR
(犘<0.01)(g5)。
2.2.3 @AÝÁÙ狀犻犳犎ڎÛI ÀìAáBï`
Tíî

ÙCêiÞ狀犻犳犎®5ëA„€óI(ë3)。
0~5cmْ,ÜÓùÙCêiÞ狀犻犳犎®5ëA©…
†€éDÒT1Óù,T5T4…†€éT36T2,T5
T4ã%غAW…†QR。5~10610~15cmÙ
’T5Óù…†€éDÒ,‘XÓùD҈¸Øº6
…†
。15~20620~25cmْT46T5ÓùêiÞ
狀犻犳犎®5ëA…†€éDÒT1,‘&15~20cmْ
T26T3D҈¸Øº6…†,20~25cmْT3
Óù…†séDÒ
,T2DÒT1ã%غ6…†。
ghAáBï_§€0~25cmْÙCêiÞ
狀犻犳犎®5qëë,êiÞc`‚ƒ,ò.G€:
ŠL0~5cmْgPÝnh…。ˆ‰=•gh,A
áBïQRÙCêiÞ狀犻犳犎 ®5ëATˆ‰]ë
n-0.779,IöWd…†QR(g5)。
2.3 š›J,-.adÏ
AáBïDÙC?9KOP:TUV~ë
4~61˜。èë4Lã,6@@ATAáBïDÜ
ْHލ:ï…†UV
,T5Óù20~25cm
ْHÞOP:…†íî

n6@`TAáB
ïD0~25cmÜْ—Þ、êiލ:©ï…†
UV

ë56ë6),ghAáBïˆ$_DÙC?
9KOPä’ïh…UV

=4 ab-.adϹl
Fig.4 Diversityindexesofbacterialcommunities
=5 cb-.adϹl
Fig.5 Diversityindexesoffungicommunities
=6 dKb-.adϹl
Fig.6 Diversityindexesofazotobactercommunities
85
!1# €?†?:AáDDE-_ÙC?9KOPTUV
`5 STefóôWJ,1fJ,Nø`€29Ï
Table5 Therelationshipsbetweenbiogasslurryvolumeandmicrobialbiomassormicrobialgeneabundance
?9K`C
SMBC
?9K`N
SMBN
16SrDNA®5ëA
16SrDNAgeneabundance
SSUrDNA®5ëA
SSUrDNAgeneabundance
狀犻犳犎 ®5ëA
狀犻犳犎geneabundance
AáBïQR
Concentrationofbiogasslurry
0.833 0.824 0.968 0.972 -0.779
  ü:g˜…†ˆ‰(犘<0.05),g˜ú…†ˆ‰(犘<0.01)
Note:indicatesignificantcorrelationatthe0.05level,indicatesignificantcorrelationatthe0.01level
3 ˆ‰
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ò}wÙC?9KcJKTUVÄAQ=}ŽÙ
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[13]。
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[45],
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[25]。

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¢9u

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nÙC&TN,TP6TK»`(<
ð

Z‘©`PCRTefAgh,AáBï[\Ù
C&»16SrDNA®5THÞ6»SSUrDNA®
5T—ÞY`…I

íî16SrDNA ®56—Þ
SSUrDNA®5ëA,I)¡»狀犻犳犎 ®5Tê
iÞT…I

狀犻犳犎 ®5ëA…†€。Coelho
?
[27]
Lsy
(犆犺犻狀犲狊犲狊狅狉犵犺狌犿)ÙC&°P,狀犻犳犎
®5ëAÀi”äd`Tíîn€

.»/0
efß#

n Ogilvie?[28]6Juraeva?[29]T/0
z°P

i”TäƒLíî狀犻犳犎 ®5TëA,.L
§i”äƒ`J‰

èéÙCêi?9KTɏ
cÙC C/N TUV[2526],ÙC C/N „‡ˆ
‰
[3031],
pH „Wˆ‰[32]。»/01BïTAá
C/Nt€,îSP“ÙCnØ_ºC,pH €
(4.36~5.12),.L§Ú"#»/0êiÞ/)
TÌ«.5

4 p‰
  0~20620~40cmْÙCTN,TP6TK
»`<ð

nÙC?9K`N6C©ÀìA
áBï`Tíîníî

ò?9K`C6NAá
BïQRú…†‡ˆ‰

g5),ˆ‰]ë=¾n
0.83360.824,‘&iCÚ`n200kg·hm-2T
W6Bï
(T5)ÙC?9K9K`N6CÝs。
T1D҈¸,0~25cmÜْT2~T5Ó
ùÙCHÞ16SrDNA®5ëA6—ÞSSUrDNA
®5ëAYú=…†íî

òÙCHÞ16SrDNA
®5ëA6—ÞSSUrDNA®5ëAAáBï
QRTú…†‡ˆ‰

ˆ‰]ë=¾n0.9686
0.972。@2,0~5cmْÙC狀犻犳犎 ®5ëAÀ
AáBï`Tíîn…†€

‘Xْ7Ls×
AAáBï
(T5$T4)2ÙC狀犻犳犎 ®5ëA’…
†€

@2AáBïQRÙCêiÞ狀犻犳犎 ®
5ëAgPnWˆ‰

IöWd…†QR

6@A
áBï`D0~25cmÜْ—Þ、êiލ:©
UV

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