Tillage System Affects Fertilizer Induced N2O Emissions Along Soil Organic Carbon Stratification

Supplement Table S1 Quantitative PCR master mix compositions and thermal profiles for the different functional genes

Target gene / Reaction mixture / Volume [µl] / Thermal profile / Reference
16S rRNA / Kapa SYBR®Fast qPCR Kit Master Mix (2X) Universal
341F (2 µM)
797R (2 µM)
PCR water
DNA - template / 5
0.4
1.2
2.4
1 / 95°C – 10 sec
61.5 – 20 sec
72°C – 20 sec / 35 cycles / modified after Nadkarni et al. (2002)
archaeal amoA
(AOA) / Kapa SYBR®Fast qPCR Kit Master Mix (2X) Universal
amo19F (5 µM)
CrenamoA616r48x (5 µM)
PCR water
DNA - template / 5
1
1
2
1 / 95°C – 15 sec
55°C – 30 sec
72°C – 30 sec / 40 cycles / modified after Towe et al. (2010)
bacterial amoA
(AOB) / Kapa SYBR®Fast qPCR Kit Master Mix (2X) Universal
amoA1F (5 µM)
amoA2R (5 µM)
BSA (10% w/v)
PCR water
DNA - template / 5
0.75
0.75
0.4
2.1
1 / 95°C – 15 sec
59.5°C – 30 sec
72°C – 30 sec / 40 cycles / modified after Towe et al. (2010)
nirK / Kapa SYBR®Fast qPCR Kit Master Mix (2X) Universal
nirK876 (2 µM)
nirK1040 (2 µM)
PCR water
DNA - template / 5
1
1
2
1 / 95°C – 10 sec
62-57°C – 20 sec
72°C – 10 sec
95°C – 15 sec
57°C – 20 sec
72°C – 10 sec / 6 cycles
35 cycles / modified after Babić et al. (2008)
nirS / Kapa SYBR®Fast qPCR Kit Master Mix (2X) Universal
nirScd3af (5 µM)
R3cd (5 µM)
PCR water
BSA
DNA - template / 5
1
1
1.6
0.4
1 / 95°C – 15 sec
58°C – 30 sec
72°C – 15 sec / 40 cycles / modified after Babić et al. (2008)
nosZ / Kapa SYBR®Fast qPCR Kit Master Mix (2X) Universal
nosZ2R (5 µM)
nosZ2F (5 µM)
PCR water
DNA - template / 5
1
1
2
1 / 95°C – 10 sec
65-60°C – 20 sec
72°C – 15 sec
95°C – 15 sec
60°C – 15 sec
72°C – 15 sec / 6 cycles
35 cycles / modified after Babić et al. (2008)

Supplement Table S2 Primers used for quantitative PCR

Target gene / Primer / Sequence 5`- 3` / Amplicon size (bp) / Reference
16S rRNA / 341F
797R / CCT ACG GGA GGC AGC AG
GGA CTA CCA GGG TAT CTA ATC CTG TT / 466 / (Muyzer et al. 1993)
(Nadkarni et al. 2002)
archaeal amoA / amo19F
CrenamoA616r48x / ATG GTC TGG CTW AGA CG
GCC ATC CAB CKR TAN GTC CA / 628 / (Leininger et al. 2006)
(Schauss et al. 2009)
bacterial amoA / amoA1F
amoA2R / GGG GTT TCT ACT GGT GGT
CCC CTC KGS AAA GCC TTC TTC / 491 / (Rotthauwe et al. 1997)
nirK / nirK876C
nirK1040 / ATY GGC GGV CAY GGC GA
GCC TCG ATC AGR TTR TGG TT / 162 / (Henry et al. 2006)
nirS / nirScd3af
R3cd / AAC GYS AAG GAR ACS GG
GAS TTC GGR TGS GTC TTG A / 413 / (Kandeler et al. 2006; Throbäck et al. 2004)
nosZ / nosZ2F
nosZ2R / CGC RAC GGC AAS AAG GTS MSS GT
CAK RTG CAK SGC RTG GCA GAA / 267 / (Henry et al. 2006)

Supplement Table S3 Pearson correlation coefficients of soil organic C (SOC) contents and functional gene abundances across all soil samples before incubation

SOC / 16S rRNA / archaeal amoA / bacterial amoA / nirK / nirS
16S / 0.88
archaeal amoA / -0.37 / 0.10
bacterial amoA / -0.20 / 0.29 / 0.98
nirK / 0.68 / 0.94 / 0.42 / 0.58
nirS / 0.88 / 0.78 / -0.26 / -0.11 / 0.68
nosZ / 0.95 / 0.73 / -0.60 / -0.44 / 0.45 / 0.68

References

Babić KH, Schauss K, Hai B, Sikora S, Redžepović S, Radl V, Schloter M (2008) Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.) Environmental Microbiology 10:2922-2930 doi:10.1111/j.1462-2920.2008.01762.x

Henry S, Bru D, Stres B, Hallet S, Philippot L (2006) Quantitative Detection of the nosZ Gene, Encoding Nitrous Oxide Reductase, and Comparison of the Abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils Appl Environ Microbiol 72:5181-5189 doi:10.1128/aem.00231-06

Kandeler E, Deiglmayr K, Tscherko D, Bru D, Philippot L (2006) Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland Appl Environ Microbiol 72:5957-5962 doi:10.1128/aem.00439-06

Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI, Schuster SC, Schleper C (2006) Archaea predominate among ammonia-oxidizing prokaryotes in soils Nature 442:806-809 doi:10.1038/nature04983

Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA Appl Environ Microbiol 59:695-700

Nadkarni MA, Martin FE, Jacques NA, Hunter N (2002) Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set Microbiology 148:257-266 doi:10.1099/00221287-148-1-257

Rotthauwe J, Witzel K, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations Appl Environ Microbiol 63:4704-4712

Schauss K et al. (2009) Dynamics and functional relevance of ammonia-oxidizing archaea in two agricultural soils Environmental Microbiology 11:446-456 doi:10.1111/j.1462-2920.2008.01783.x

Throbäck IN, Enwall K, Jarvis Å, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE FEMS Microbiology Ecology 49:401-417 doi:10.1016/j.femsec.2004.04.011

Towe S, Albert A, Kleineidam K, Brankatschk R, Dumig A, Welzl G, Munch JC, Zeyer J, Schloter M (2010) Abundance of Microbes Involved in Nitrogen Transformation in the Rhizosphere of Leucanthemopsis alpina (L.) Heywood Grown in Soils from Different Sites of the Damma Glacier Forefield Microbial Ecology 60:762-770 doi:10.1007/s00248-010-9695-5