Appendix A Supplementary data
Evaluation of a crop rotation with biological inhibition potential to avoid N
2O emissions in comparison with synthetic nitrification inhibition
Adrián Bozal-Leorri
1,*, Mario Corrochano-Monsalve
1, Luis M. Arregui
2, Pedro M. Aparicio- Tejo
3, Carmen González-Murua
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Department of Plant Biology and Ecology, University of the Basque Country (UPV/EHU), Bilbao 48940, Spain
2
Institute for Innovation and Sustainable Development in Food Chain (ISFOOD), Public University of Navarre, Pamplona 31006, Spain
3
Institute for Multidisciplinary Research in Applied Biology (IMAB), Public University of Navarre, Pamplona 31006, Spain
---
*Corresponding author. E-mail: [email protected] (A. Bozal-Leorri).
FIGURES
Fig. S1 Daily precipitation (blue bars) and mean air temperature (red line) of growing season 2019/2020.
Fig. S2 Evolution of soil temperature (0-10 cm) (red line) and soil Water Filled Pore Space (0-30 cm) (blue line) during N
2O emissions measurements in winter wheat experiment.
2
TABLE
Table S1 Primers pairs and thermal conditions used in real-time qPCR.
Target group
Primer
name Sequence Thermal profile Length
(bp) References
Bacterial 16S rRNA
341F 5'-CCTACGGGAGGCAGCAG-3' 95℃ for 2 min (
×
1 cycle)95℃ for 15 sec, 60℃ for 30 sec, 72℃ for 30 sec, 80℃ for 30 sec (
×
40 cycles)174 Lopez-Gutiérrez et al. (2004)
534R 5'-ATTACCGCGGCTGCTGGCA-3'
Bacterial amoA
amoA1F 5'-GGGGTTTCTACTGGTGGT-3' 95℃ for 2 min (
×
1 cycle)95℃ for 15 sec, 54℃ for 60 sec, 72℃ for 60 sec (
×
40 cycles)491 Rotthauwe et al. (1997)
amoA2R 5'-CCCTCKGSAAAGCCTTCTTC-3'
nirK
NirK 876 5´-ATYGGCGGVCAYGGCGA-3´
95℃ for 2 min (
×
1 cycle)95℃ for 15 sec, 63℃ for 30 sec (-1
°C /cycle), 72℃ for 30 sec, 80℃ for 15 sec (
×
6 cycles)95℃ for 15 sec, 58℃ for 30 sec, 72℃ for 30 sec, 80℃ for 30sec (
×
40cycles)
165 Henry et al. (2004)
NirK1040 5´-GCCTCGATCAGRTTRTGGTT-3´
nirS
cd3aF 5´-GTSAACGTSAAGGARACSGG-3 ´ 95℃ for 2 min (
×
1 cycle) 95℃ for 45 sec, 55℃ for 45 sec, 72℃ for 45 sec, 85℃ for 20 sec (×
40 cycles)410
Michotey et al. (2000)
R3cd 5´-GASTTCGGRTGSGTCTTGA-3´ Throbäck et al. (2004)
nosZI
nosZ-F 5'-CGCRACGGCAASAAGGTSMSSGT-3' 95℃ for 2 min (
×
1 cycle) 95℃ for 15 sec , 63℃ for 30 sec (- 1℃/cycle), 72℃ for 30 sec, 80℃ for15 sec (
×
6 cycles)95℃ for 15 sec, 60℃ for 30 sec, 72℃ for 30sec, 80℃ for 30 sec (
×
40267 Henry et al. (2006)
nosZ-R 5'-CAKRTGCAKSGCRTGGCAGAA-3'
4
cycles) nosZII
nosZ-II-F 5´-CTIGGICCIYTKCAYAC-3´ 95℃ for 2 min (
×
1 cycle) 95℃ for 30 sec, 54 °C for 30 sec,72℃ for 40 sec, 85℃ for 15 sec (
×
40 cycles)698 Jones et al. (2013)
nosZ-II-R 5´-GCIGARCARAAITCBGTRC-3´
Table S2 Wheat grain yield, yield components, grain protein, N yield scaled N
2O emissions (YSNE) and Nitrogen Use Efficiency (NUE).
Grain yield
(kg/ha ) Spikes/m2 1000 grains DW
(g) Grains/spike Grain protein (%)
YSNE (g N2O-N/
kg N uptake)
NUE (kg dry matter/kg N)
Fallow-wheat
Control 5120 ± 520 b 313 ± 10 b 40.3 ± 1.0 a 39.9 ± 2.8 a 7.1 ± 0.2 a 1.7 ± 0.3 b
AS 6616 ± 452 a 452 ± 30 a 40.4 ± 0.5 a 36.9 ± 3.6 a 7.0 ± 0.4 a 4.5 ± 0.6 a 22.0 ±
6.8 a
AS+DMPP 6133 ± 271 a 395 ± 22 a 39.7 ± 1.1 a 42.0 ± 5.8 a 6.7 ± 0.3 a 1.2 ± 0.2 b 22.2 ±
5.1 a
Sorghum- wheat
Control 3860 ± 103 B
* 252 ± 18 B
* 38.5 ± 0.4 B 40.0 ± 4.4 A 7.0 ± 0.2 A 1.7 ± 0.1 B
AS 6230 ± 362 A 439 ± 21 A 39.9 ± 0.4 A 36.7 ± 4.2 A 7.3 ± 0.2 A 8.7 ± 1.0 A
*
26.3 ±
4.0 A
AS+DMPP 6543 ± 347 A 429 ± 35 A 39.7 ± 0.3 A 40.0 ± 3.6 A 7.0 ± 0.1 A 1.2 ± 0.4 B 29.8 ±
3.7 A
Control: control without fertilisation; AS: fertilised with ammonium sulphate; AS+DMPP: fertilised with ammonium sulphate + 3,4 dimethylpyrazole phosphate. Significant differences between treatments of “Fallow-wheat” rotation are marked with lowercase letters. Significant differences between treatments of “Sorghum-wheat” rotation are marked with capital letters. For both ANOVA, the Duncan Test was used (p <0.05; n = 4). The Mann- Whitney U test was used for the comparison between crop rotations within the same fertilization treatment. Significant differences at p <0.05 are marked with an asterisk (*).
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