Experimental sitesThe field experiment was conducted from the 21 May 2012 to 20 April 2013 in a paddock located near Hamilton (142o07'E, 37o82’S) in south west Victoria; on a Ferric-Eutrophic Brown Chromosol soil (Isbell 2003). Topsoil (0-35cm) clay content was 15-20%, thereafter abruptly increasing to 70% at 40 cm depth, and remaining at 70% throughout the rest of the soil profile.Paddock historyPrior to the establishment of the experiment, the paddock consisted of a long-term (50 years) mixed sward of subterranean clover (Trifolium subterraneum L.) and perennial grass pasture until 2004, since periodically cropped with winter wheat (Triticum aestivum L.) and canola (Brassica napus). On 1 September, 5 October and 14 December 2011, a tank mix of glyphosate (1080 g of a.i./ha) and Carfentrazone-ethyl (18 g of a.i./ha) was sprayed on each occasion to impose a chemical fallow proceeded by deep ripping on 13 January and again on 7 March, followed by power harrowing on 15 March, before forming raised beds on 21 March 2012. Beds were 1.35 m wide and adjacent furrows 15 cm deep by 35 cm wide. Wheat cv. Bolac was planted in 2012, followed by canola cv. Stingray in 2013, and returned to wheat in 2014.Experimental designThe experiment comprised a completely randomised block design with ten treatments replicated five times. Plots were 10 m long by 3.4 m wide; or two raised beds with one dividing furrow per plot. The treatments included a 0N experimental control (0N), four additional rates of N fertiliser (TD10N@Z31, TD35N@Z31, TD85N@Z31 and TD185N@Z31) top-dressed at the first node (Z31) growth stage of wheat (Zadocks et al. 1974); three more rates of urea N with additional foliar Cu fertiliser (0N+Cu@Z25&31,TD35N+Cu@Z25&31 and TD85N+Cu@Z25&31); a further treatment involving DMPP (3,4-Dimethylpyrazole phosphate, Entec®) coated urea (DMPP85N@Z00) deep banded at sowing; and finally one deep banded urea (DB85N@Z00) treatment imposed at sowing. All treatments received a basal application of 15 kg of P/ha at sowing and all treatments except the 0N control received a basal application of 15 kg of N/ha as sowing (Table 1). The DB85N@Z00 and DMPP85N@Z00) treatments involved deep banding fertiliser to a depth of 10 cm, below the intended wheat seeding depth the day before planting. Table 1.Treatments imposed at the Hamilton field experiment, in south west Victoria.Treatment Code Basal Basal P Basal N DB TD0N DSP 15 0 0 0TD10N@Z25 DAP 15 15 0 10TD35N@Z25 DAP 15 15 0 35TD85N@Z25 DAP 15 15 0 85TD185N@Z25 DAP 15 15 0 185DMPP85N@Z00 DAP 15 15 85 0DB85N@Z00 DAP 15 15 85 00N+Cu@Z25&31 DSP 15 0 0 0TD35N+Cu@Z25&31 DAP 15 0 0 35TD85N+Cu@Z25&31 DAP 15 0 0 85DSP = Double Superphosphate fertiliserDAP = Di-Ammonium Phosphate fertiliserDB = Deep banded at 10 cm depth below the seed at sowing (Z00)TD = Top-dressed at the first node growth stage of wheat (Z31)Baseline soil chemical and physical propertiesBefore experimentation, soil organic C, total soil N, soil P and exchangeable aluminium levels generally declined with depth, and conversely soil pH, exchangeable sodium and bulk density increased with depth (Table 2). Electrical conductivity, sulphur and potassium initially decreased with depth and then increased in the deeper layers. In the topsoil layer (0 – 10 cm) of the furrow, all soil parameters were lower in comparison to the adjacent beds, with the exception of exchangeable aluminium and bulk density (Table 2). Table 2. Soil chemical and physical properties at the Hamilton field experiment, in south west Victoria*.Soil Organic Total Soil Electrical Exchangeable Exchangeable SulphurF Phosphorus Potassium Bulkdepth carbonA nitrogenB pHC conductivityD aluminiumE sodiumE colwellG colwellG densityH(cm) (%) (%) (CaCl2) (dS/m) (%) (%) (mg/kg) (mg/kg) (mg/kg) (g/cm3)Bed top0 - 10 2.82 (± 0.10) 0.28 (± 0.01) 5.68 (± 0.17) 0.18 (± 0.02) 0.21 (± 0.09) 1.66 (± 0.10) 39.10 (± 2.71) 62.00 (± 4.34) 155.40 (± 10.54) 1.2010 - 20 2.16 (± 0.09) 0.22 (± 0.01) 5.34 (± 0.05) 0.08 (± 0.01) 0.41 (± 0.08) 1.54 (± 0.10) 13.20 (± 1.21) 36.20 (± 2.03) 72.40 (± 6.61) 1.2520 - 30 1.01 (± 0.10) 0.10 (± 0.00) 5.50 (± 0.07) 0.04 (± 0.00) 0.39 (± 0.07) 2.41 (± 0.08) 13.30 (± 2.23) 13.60 (± 0.60) 49.20 (± 3.14) 1.3730 - 40 0.59 (± 0.06) 0.05 (± 0.01) 5.96 (± 0.05) 0.04 (± 0.01) 0.80 (± 0.08) 3.93 (± 0.11) 13.20 (± 1.42) 7.40 (± 1.72) 51.00 (± 3.99) 1.3940 - 60 0.59 (± 0.11) 0.05 (± 0.01) 6.12 (± 0.04) 0.06 (± 0.01) 1.18 (± 0.12) 5.87 (± 0.43) 21.08 (± 3.20) 2.80 (± 0.20) 65.20 (± 5.27) 1.5560 - 80 0.38 (± 0.03) 0.04 (± 0.00) 6.04 (± 0.02) 0.07 (± 0.00) 1.10 (± 0.13) 7.57 (± 0.58) 33.06 (± 2.55) 2.25 (± 0.22) 69.00 (± 3.71) 1.6880 - 100 0.28 (± 0.04) 0.03 (± 0.00) 6.10 (± 0.03) 0.09 (± 0.00) 0.93 (± 0.08) 9.03 (± 0.45) 36.68 (± 3.24) 2.33 (± 0.26) 71.80 (± 4.24) 1.70furrow#0 - 10 2.39 (± 0.13) 0.24 (± 0.01) 5.70 (± 0.08) 0.11 (± 0.01) 0.19 (± 0.04) 1.69 (± 0.06) 26.16 (± 1.90) 50.60 (± 1.50) 172.80 (± 8.91) 1.3*Values represent the mean of the five replicates, numbers in brackets ±SE. #Separate 0-10cm samples were collected from the bed top and adjacent furrow. AMeasured by Walkley and Black (1934) method. BMeasured by combustion of air dry soils using LECO combustion analyser. CMeasured in 0.01M CaCl2 solution at a 1:5 soil to extract ratio using a glass electrode. DMeasured in water using a probe and a1:5 soil to extract ratio. EMeasured in 0.1M NH4Cl/0.1M BaCl2 at a 1:10 soil to extract ratio for two hours, before concentrations determined by Inductively Coupled Plasma. FMeasured by Colwell (1965) and Raymond and Higginson (1992) methods. GMeasured by Blair et al. (1991) method. Hspecified elsewhere in the Materials and Methods.Climatic and soil temperature measurementsAn automated tipping bucket rain gauge (Hastings Dataloggers, Port Macquarie, Australia, www.hdl.com.au) installed in close proximity to the experimental site measured hourly rainfall. Hourly topsoil water was monitored by theta probes (Theta-Probe MK2x, Delta-T Devices Ltd, Burwell England) installed to 6 cm depth, and hourly topsoil (0-10cm) temperature by FT100 temperature probes (Hastings Dataloggers, Port Macquarie, Australia, www.hdl.com.au). Probes (theta and soil temperature) were installed in the TD10N@Z31 treatments; and were placed on top of the raised bed and in the middle of the adjacent furrow.Crop managementUrea was deep banded through a cone seeder to the DB85N@Z00 and DMPP85N@Z00 treatments on 7th of May 2014. A tank mix of Glyphosate (1080 g of a.i./ha) and Pyroxasulfone (100 g of a.i./ha) was sprayed shortly before sowing wheat cv. Bolac at 85 kg/ha on the 7th of May 2014. Either a basal application of N and P was applied as DAP (15 kg/ha of N and 15 kg/ha of P) or DSP (15 kg/ha of P) with the seed depending on the treatment, and the fertiliser was treated with Flutriafol (125 g of a.i./ha) to combat future threats of stripe rust (Puccinia striiformis). Plots were sown using a cone seeder with knife points and press wheels spaced 15 cm apart. On the 2nd of July 2014, MCPA and Diflufenican (188 g and 19 g of a.i./ha, respectively) were sprayed to control volunteer canola and germinating capeweed (Arctotheca calendula) and wild radish (Raphanus raphanistrum) populations. Several applications of Alpha-Cypermethrin (10 g of a.i./ha) and Omethoate (29 g of a.i./ha) were made to control aphids (Rhopalosiphum padi and (Rhopalosiphum maidis) and Red-Legged Earthmite (Halotydeus destructor) on the 6th and 19th of May, 2nd of July and 9th of August 2014. Propiconazole (125 g of a.i./ha) fungicide was applied to prevent outbreaks of Septoria Tritici Blotch (Mycosphaerella graminicola) on the 9th of August and 26th of September 2014.Treatments allocated to topdressing (TD10N@Z25, TD35N@Z25, TD85N@Z25, TD185N@Z25, TD35N+Cu@Z25&31 and TD85N+Cu@Z25&31) received additional urea on the 18th of July, coinciding with the mid to late tilling stage of wheat growth. The 0N+Cu@Z25&31, TD35N+Cu@Z25&31 and TD85N+Cu@Z25&31 treatments received two application of Copper foliar fertiliser (2.5 L a.i./ha) on the 22nd of July and 14th of August coinciding with the mid till to first node growth stage of wheat. Fertiliser was not applied to the adjacent furrows in all treatments. Stubble remained standing on the site over the fallow period.Soil sample collection for chemical analysis and bulk densityFive deep soil cores (internal diameter 42 mm) were randomly collected from the beds in each replicate on 24 April 2014, and three cores randomly collected from the 0N, DB85N@Z00, DMPP85N@Z00, TD35N@Z31 and TD85N@Z31 plots on 19 December 2014. On each occasion the cores were divided into 10 cm increments to 40 cm depth, and thereafter in 20 cm increments. Four of the five cores collected in April 2014 and all the cores collected in December 2014, were combined for each layer within each replicate or plot. Samples were then oven dried at 40°C for 48 h and passed through a 2 mm sieve in preparation for chemical analysis. The remaining core collected from each replicate in May 2012 were weighed and oven dried at 105oC for 48 h and weighed again to determine gravimetric water, bulk density and or volumetric water.Surface soil mineral N (NH4+ and NO3-) was measured monthly throughout the study; with more frequent samples taken before and after N fertiliser applications. On each occasion between 12 and 15 soil cores (internal diameter 20 mm) were randomly collected to a depth of 10 cm, initially from the 0N and DB85N@Z00 plots, then later additional DMPP85N@Z00, TD35N@Z31 and TD85N@Z31 treatments. Within each plot separate soil samples were taken from the beds and from the adjacent furrows. Samples were oven dried at 40oC for 48 h, and passed through a 2 mm sieve in preparation for soil mineral N analysis.Crop measurementsCrop emergence was measured at the second leaf stage (Z12) of wheat growth by counting plants on both sides of a 0.5 m stick, randomly placed 10 times within each plot. Wheat biomass was measured at first node (Z31), anthesis (Z65) and maturity (Z93) on 8 August 2014, 17 October 2014 and 16 December 2014 respectively, by cutting two random locations of 1 m row of crop. On each occasion crop was cut at ground level and bulked within each plot, subsamples retained and oven dried at 65oC until constant weight reached. After drying, spike density was measured by counting fully emerged wheat ears collected from anthesis (Z65) biomass samples, and maturity biomass (Z93) samples were threshed to separate grain from straw.Wheat grain yield was measured by mechanically harvesting each plot. A sub-sample of grain was retained to assess grain quality. Wheat grain protein was calculated by multiplying grain N concentration by 5.7 (Halvorson et al. 2004). Grain weight was determined by the mass of grain within a hectolitre (hL) volume; then passed through a 2 mm sieve to quantify grain screenings.N2O gas sample collectionN2O gas concentrations were measured from 24 April 2014 to 16 December 2014, taken on a fortnightly to monthly frequency during the study period, except when N fertiliser was applied at sowing (Z00) and first node of wheat growth (Z31), when N2O gas was measured 1 day, 3 days and 7 days after application from the beds.N2O gas was collected manually from the beds of the 0N, DB85N@Z00, DMPP85N@Z00, TD35N@Z31 and TD85N@Z31 treatments, and from the furrows of the 0N and DB85N@Z00 treatments using the static chamber methodology described by Harris et al. (2013). Two metal trough bases were placed in close proximity within each plot and two more in the adjacent furrows of the 0N and DB85N@Z00 treatments only, accompanied by raised wooden platforms to minimise crop damage and soil disturbance when gas sampling. At sampling chambers were placed into the ground level metal troughs filled with water where fluxes were measured between 10 am and 2 pm. Samples were collected by syringe at 0, 20, 40 and 60 minutes after lid emplacement. Twenty mL air samples were injected into 12 ml evacuated exetainers (Labco Limited, High Wycombe, UK) and posted to the University of Melbourne, Parkville Campus for analysis by gas chromatograph. Tinytag plus 2 temperature data loggers (Hastings Dataloggers, Port Macquarie, Australia, www.hdl.com.au) were placed inside one chamber in each replicate to monitor changes in air temperature during gas sampling.Calibration of the Theta probe and conversion to water filled pore space (WFPS)When topsoil samples were collected for soil mineral N analysis, a subsample was retained, weighed and oven dried at 105oC for 48 h and weighed again to determine gravimetric water. A regression analysis determined an equation, used to convert theta probe data to volumetric water. Calibration equations included:Beds:Volumetric soil water = (0.0097 x water content) + 0.0115 (R2 = 0.96)Furrows:Volumetric soil water = (0.0072 x water content) + 0.0296 (R2 = 0.96)Water filled pore space was then determined by dividing volumetric water content by total porosity (Linn and Doran 1984).Chemical and data analysisSoil NO3- and NH4+ analysis involved soils extracted with 1M of KCl solution for 1 h at 25C; the resulting solution was then measured on a Lachat Flow Injection Analyzer (Searle 1984). Grain and plant N concentrations were measured using a LECO CNS2000 analyser apparatus. Gas samples were analysed by a fully automated Gas Chromatograph (Agilent 7890A, Agilent Technologies Inc. Wilmington, USA) equipped with a micro electron capture detector to quantify N2O (N2O(g)) concentration and then converted to gas density (N(g)) by:N(g) = N2O(g) x (P x 2Mw)/(R x T)where P is atmospheric standard air pressure of 101.31 kPa, Mw is the molecular weight of N, R is the universal gas constant (8.314 j K-1 mol-1), and T is chamber air temperature (Kelvin). Gas density was then adjusted for chamber volume. Fluxes were calculated from the linear increase in gas density in the chamber headspace with time; flux rates with a regression coefficient (r2) of <0.80 were discarded (Barton et al. 2008).ReferencesBarton L, Kiese R, Gatter D, Butterbach-Bahl K, Buck R, Hinz C, Murphy DV (2008) Nitrous oxide emissions from a cropped soil in a semi-arid climate. Global Change Biology 14, 177–192.Blair GJ, Chinoim N, Lefroy RDB, Anderson GC, Crocker GJ (1991). A soil sulfur test for pastures and crops. Australian Journal of Soil Research 29, 619-626.Colwell JD (1965). 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