Winter wheat cultivars and nitrogen (N) fertilization—Effects on root growth, N uptake efficiency and N use efficiency

Europ. J. Agronomy 68 (2015) 38–49

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Winter wheat cultivars and nitrogen (N) fertilization—Effects on root growth, N uptake efficiency and N use efficiency Irene Skovby Rasmussen ∗ , Dorte Bodin Dresbøll, Kristian Thorup-Kristensen Department of Plant and Environmental Sciences, University of Copenhagen, Højbakkegaard Allé 13, 2630 Taastrup, Denmark

a r t i c l e

i n f o

Article history: Received 19 December 2014 Received in revised form 1 April 2015 Accepted 22 April 2015 Keywords: Root depth Root density Deep soil layers Soil N depletion NUE NUpE

a b s t r a c t One way to reduce nitrate leaching losses from agricultural land is to increase crop nitrogen uptake efficiency (NUpE). In this aspect, root growth is an essential parameter, as more and deeper roots may improve the uptake from deeper soil layers and reduce nitrate leaching. This study examined the root growth, soil N depletion and yields of modern, commercial winter wheat (Triticum aestivum L.) cultivars in a two-year field experiment conducted on sandy loam soils. The effects of N fertilization on root growth and inorganic soil N utilization were quantified. In order to obtain data from the entire winter wheat rooting zone, the measurements were conducted to 2.3 m soil depth. Root growth was studied by means of minirhizotrons. Parallel to the belowground measurements, the effects on aboveground biomass and N uptake were measured. In the two experimental years the average maximum root depths were 1.1 and 1.5 m, respectively, and the average root depth penetration rates were 0.7 and 1.0 mm ◦ C day−1 , respectively. N fertilization affected root density, which increased at least up to an application of 150 kg N ha−1 . The effect on root density was mostly seen in soil layers below 0.5 m. N fertilization did not appear to affect root depth. There were root growth differences among the cultivars, though not strongly pronounced. The cultivar Hereford showed tendencies to higher root densities and deeper root growth, and this trend was correlated with a tendency to increased subsoil N depletion. Also, Hereford showed higher N use efficiency (NUE) compared to the other cultivars, as it produced more grain per N supply. Under the existing experimental conditions, a spring application up to 150 kg N ha−1 did not increase the amount of nitrate left in the soil at harvest. In contrast, by an increase of N fertilization from 150 to 250 kg N ha−1 , on average 36 % of the extra 100 kg N ha−1 was left in the soil. With a further increase from 250 to 350 kg N ha−1 , up to 90% of the extra N was left in the soil. The soil N increase at high N fertilization was most profound in the upper soil layer but also significant in the subsoil. © 2015 Elsevier B.V. All rights reserved.

1. Introduction It is necessary to increase nutrient use efficiency of common crops in order to meet the future need for food, feed and fuel without excessive resource use or nutrient losses to the environment. Wheat (Triticum aestivum L.) is the main field crop in many temperate areas (FAO, 2014) and efficient use of nitrogen (N) fertilizer is of high concern, because it is associated with eutrophication of fresh water and marine ecosystems, with greenhouse gas emissions and with high levels of energy use. Root growth is an essential parameter for crop N uptake efficiency, as more and deeper roots may improve the uptake from

∗ Corresponding author. Tel.: +45 35333561; fax: +45 35329577. E-mail address: [email protected] (I.S. Rasmussen). http://dx.doi.org/10.1016/j.eja.2015.04.003 1161-0301/© 2015 Elsevier B.V. All rights reserved.

deeper soil layers and reduce nitrate leaching losses to the environment (Gastal and Lemaire, 2002; King et al., 2003). N fertilization will in itself have an effect on root growth in ways we only partly understand. It will affect overall plant growth and N status, and thereby also root growth, but soil N content will also affect root growth more directly leading to a local increase in root growth (Forde, 2002). Studies of the relationship between root growth and N availability have shown a general tendency for plant roots to proliferate in nutrient rich zones, while root growth is suppressed in zones of low nutrient supply (Robinson, 1994). Presumably, this response enables plants to partially compensate for non-uniform supplies of nutrients (Robinson, 1994). While local root proliferation can cause greater exploitation of immobile resources such as phosphate, the adaptive function of local root proliferations is less clear for mobile soil resources like nitrate. Increased root proliferation in N-rich zones may give

I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

single plants a competitive advantage against its neighbours, but it will have a limited effect on overall crop N capture (Robinson, 1996; Robinson et al., 1999). In regard to N capture, deeper root growth is likely to be more important than increased root density, as deeper roots give the crop access to a larger volume of soil and the resources within this. Field studies on winter wheat have shown root growth to 1.5–2.0 m soil depth (Gregory et al., 1978; Kuhlmann et al., 1989; Anderson et al., 1998; Sauer et al., 2002; Kirkegaard and Lilley, 2007). In general, field studies have reported agreement between root depth observations and N depletion from deep soil layers (Kristensen and Thorup-Kristensen, 2004; Haberle et al., 2006; Thorup-Kristensen et al., 2009). Less is known about the influence of N fertilizer on overall root growth and N uptake from deep soil layers, and the existing findings seem ambiguous, though it has been found in field studies that winter wheat root densities increase when N fertilizer is applied (Barraclough et al., 1989). Increased root growth in deep soil layers could be a response to higher subsoil N availability due to nitrate leaching (Gao et al., 1998). Consistent with this, higher N availability in deep soil layers under field vegetable crops resulted in greater root depth penetration rates and increased root frequency in deep soil layers (Kristensen and Thorup-Kristensen, 2007). However, increased root growth and deep rooting at high N levels could also simply be due to stronger aboveground plant growth leading to enhanced root growth. On the other hand, other studies of different plant species indicate that higher soil N availability does not significantly increase root depth and density (Robinson, 1994; Gabrielle et al., 1998; Svoboda and Haberle, 2006). It has even been shown in field studies that a high fertilizer rate of 200 kg N ha−1 can reduce winter wheat root depth as well as density in the subsoil (Svoboda and Haberle, 2006). All in all, the effect of N fertilizer on root growth in deep soil layers is not clear, and the possibility of an optimum fertilizer level for deep root development does not seem to have been studied. Using genotypic variation in deep root development is another possible way to improve deep rooting and N uptake. Studies have indicated that significant variation exist among crop cultivars in this trait (Hoad et al., 2001; Ford et al., 2006; Ytting et al., 2014). It has been shown in recent studies that such variation in deep rooting also lead to significant differences in the extraction of water from deeper soil layers (Ober et al., 2014). Similar effects of N have been shown when comparing crop species with different rooting depth (Kristensen and Thorup-Kristensen, 2004), but they have not yet been documented among cultivars within a crop species. While deep rooting allows wheat to take up N from the entire root zone including the deepest parts (Kristensen and ThorupKristensen, 2004), it is less clear how the actual uptake from the subsoil is affected by N fertilization of the crops. Field studies of winter wheat have indicated a relation between N fertilization and N depletion from deep soil layers. Decreasing the N supply to the topsoil was shown to increase N uptake from the subsoil (Kuhlmann et al., 1989). In accordance with this, Haberle et al. (2006) reported delayed and reduced utilization of N from deep soil layers at high fertilization rates of 200 kg N ha−1 in winter wheat. The reasons for the relationship between topsoil N fertilization and crop uptake of N from the subsoil are illustrated by the model simulation study by Pedersen et al. (2010). The roots reach the subsoil layers late during crop development and never develop as high densities as in the top soil, all in all delaying the N uptake from the subsoil. If the N supply from fertilizer added to the upper soil layers is sufficient to cover plant N demand, it will decrease the N uptake rate of the entire root system. In this way, N depletion in deep soil layers is not only influenced by root growth, but is also affected by the total plant N status (Thorup-Kristensen and Sørensen, 1999; Pedersen et al., 2010).

39

Table 1 Clay, silt, sand content and chemical composition of the soil to a depth of 2.3 m. The values are averaged for the two experimental fields. Depth

Clay

(m)

(%)

0–0.5 0.5–1.0 1.0–1.5 1.5–2.0 2.0–2.3

15.5 20.3 19.5 18.5 19.0

Silt

Sand

pH

P

K

Mg

(mg kg−1 ) 13.8 14.8 16.0 18.0 19.8

69.0 64.5 64.5 63.3 61.8

6.6 6.9 7.3 7.5 7.6

26.8 15.3 8.0 5.8 4.8

117.5 59.8 48.8 49.3 54.0

C (%)

45.8 57.8 66.8 58.5 54.3

1.0 0.5 0.3 0.2 0.1

Currently, there is limited data available showing soil N distribution in the entire winter wheat root zone and how the N distribution is affected by N fertilization. Studies of N dynamics and root growth in deep soil layers could increase the understanding of crop utilization of available N and help improving N management in wheat cropping systems. This study examined how N fertilization and choice of winter wheat cultivar affects root growth, utilization of inorganic N from deep soil layers and N efficiency. Experiments were carried out with different N fertilizer quantities in the range from low to high N supply compared to crop demand. The main objectives were to quantify root growth patterns, soil N depletion, N uptake efficiency (NUpE) and N use efficiency (NUE) as affected by N fertilizer levels in different winter wheat cultivars. Studies were conducted to 2.3 m soil depth, in order to acquire data from the entire winter wheat root zone. It was hypothesized that (I) wheat root density and root depth are affected by N fertilizer rate, (II) there are cultivar differences in root growth, (III) increased N fertilization lead to increased levels of nitrate N residues in the soil at wheat harvest, also in the subsoil, and (IV) cultivars with deep rooting deplete subsoil nitrate better than others. 2. Materials and methods 2.1. Field site and experimental design The study was a two-year field experiment with modern, commercial winter wheat (T. aestivum L.) cultivars treated with different N fertilizer levels to study effects on root growth, soil N, aboveground biomass, crop N and yield. The experiment was carried out in the seasons 2011–12 (exp. 1) and 2012–13 (exp. 2) in two separate, conventionally grown fields at the University of Copenhagen, Department of Plant and Environmental Sciences, in Taastrup, Denmark (55◦ 40 N; 12◦ 18 E). The soil (Table 1) is an Agrudalf soil classified as sandy loam according to the ISSS classification. Temperature data (Fig. 1) was obtained from a meteorological station located less than 700 m from the experimental fields, and precipitation data was obtained from meteorological stations located less than 15 km from the experimental fields. Accumulated precipitation from 1 August 2011 until 31 July 2012 was 611 mm, and from 1 August 2012 until 31 July 2013 it was 459 mm. The experiment was placed in a randomized complete block design with three replicates and 11 treatments within each block. The plot size was 3 × 10 m. The previous crop was oat (Avena sativa L.) in exp. 1 and winter barley (Hordeum vulgare L.) in exp. 2. Straw from the previous crop was removed from the field before ploughing. The winter wheat cultivars Hereford and Cordiale were studied in both experimental years, whereas Genius and JB Asano were included in exp. 1, and Tabasco and Tuareg were included in exp. 2. Due to results found in concurrent root studies on winter wheat cultivars, it was decided to replace Genius and JB Asano with Tabasco and Tuareg in the second year of the experiment. Hereford is classified as fodder wheat, and the others are bread wheat cultivars,

I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

Average temperature (C)

30

10 days average air temperature

150

Monthly precipitation

130 110

20

90 10

70

Precipitation (mm)

40

50 0

30 10

-10 May Aug 2011

Nov

Feb May Aug 2012

Nov

Feb May 2013

Aug

Fig. 1. Weather data for the the experimental period, exp. 1 from September 2011 until August 2012 and exp. 2 from September 2012 until August 2013. The figure shows monthly precipitation (mm) and ten days average air temperature (◦ C).

though Tabasco is classified between fodder and bread as biscuit wheat. In spring, the cultivar Hereford was treated with 20, 85, 150, 250 and 350 kg N ha−1 of inorganic N fertilizer (50% NO3 − , 50% NH4 + ). The other cultivars were treated with 85 and 250 kg N ha−1 . In the following, the treatments are referred to as 20 N, 85 N, 150 N, 250 N and 350 N, respectively. Treatments higher than 150 N were divided in two applications of 150 and 100/200 kg N ha−1 . Base dressings of phosphorus and potassium were applied in spring as well. Dates and details for crop establishment, fertilizer treatments, pesticide treatments and data collections are shown in Table 2. In exp. 1, herbicides were applied once in autumn and once in spring, and fungicides were applied once in spring. In exp. 2, herbicides were applied twice in autumn, fungicides were applied once in spring and once in summer, and insecticides once in summer. These treatments effectively controlled weeds, pathogens and pests, except for some problems with grass weeds during the early season of the first year. 2.2. Root measurements Root measurements were conducted six times during the growth season: in November before winter (BBCH growth stage 13–21), in December (BBCH 23–26), in March before fertilization (BBCH 24–26), in April/May after fertilization (BBCH 31–34), in June at anthesis (BBCH 62–65) and in late July at grain ripening (BBCH 85–88). The actual measurement dates are presented in Table 2. Root depth and intensity was measured using minirhizotron tubes of 70 mm outer and 60 mm inner diameter and a total length of 3 m. In exp. 1, the tubes were made of glass, and in exp. 2 transparent plastic tubes (PMMA) were used. Two minirhizotrons were installed in every plot shortly after sowing. The minirhizotrons were inserted at an angle of 30◦ from vertical, reaching a depth of approximately 2.5 m. The aboveground 0.15–0.20 m of the tubes were covered in dark plastic to prevent light intake. To ensure that only winter wheat roots would be observed, the areas above the minirhizotrons were weeded by hand when required. A web camera was used to take pictures of the roots at the interface between the upper surface of the minirhizotron and the soil. In the photos one squared pixel represented a side length of 0.042 mm. Two photos, each covering an area of 0.02 m × 0.025 m, were taken for each 0.05 m along the left and right sides of the inner, upper surface of the minirhizotron. Each photo was analysed in a counting grid consisting of 0.005 m × 0.005 m squares with a total of 0.16 m grid line. Root intensity in each photo was measured as the total number of root

crossings on the grid line. This was used to calculate root intensity as the number of root intersections per meter grid line (intersection m−1 ) in the specific soil depth of the photo. Since the minirhizotron tubes were inserted at an angle of 30◦ from vertical, the specific soil depth of the photo was calculated as cos (30◦ ) × tube depth of the photo. The values from both minirhizotrons in a plot were averaged to one value in each 0.5 m depth interval before statistical analysis (n = 3). This single value thereby represented root observations made across a total grid line length of 6.4 m. Root depth was registered as the deepest root observed from the two photo rows on each minirhizotron. Before statistical analysis, the maximum root depths measured in the two tubes in a plot were averaged to one value for each plot (n = 3), and root depth was then calculated and presented as average maximum root depth across replicates. Root depth penetration rate (mm ◦ C day−1 ) was estimated from the slope of the regression line of average maximum root depth vs. accumulated average daily temperature assuming a base temperature of 0 ◦ C (Barraclough and Leigh, 1984). The slope of the regression line was calculated from average maximum root depths measured from the period when roots were first observed (December in exp. 1 and November in exp. 2, respectively) and until anthesis in June, when depth penetration ceased.

2.3. Biomass and soil sampling Plant material was sampled five times during the growth season: in November before winter (BBCH 13–21), in March before fertilization (BBCH 24–26), in April/May after fertilization (BBCH 31–34), in June at anthesis (BBCH 62–65) and in August at grain maturity (BBCH 85–88). The actual measurement dates are presented in Table 2. In each plot an area of 0.5 m2 gathered from two subplots of 0.25 m2 was harvested by cutting the wheat crop at ground level. The subplots were placed with a distance of approximately 1 m. In autumn and early spring the plant material was washed to remove soil. At the fourth and final harvest, the sample area was 1 m2 gathered from four subplots of 0.25 m2 and the plant material was separated in grain and other aboveground biomass. At each sample occasion the plant material was dried at 80 ◦ C for 24 h, weighed, milled and analysed for total N content using an Organic Elemental Analyzer, Flash 2000. All values are expressed as kg N ha−1 . The contents of nitrate N in the soil were measured at five occasions during each season: in August before sowing, in November before winter (BBCH 13–21), in March/April before fertilization (BBCH 24–26), in June after anthesis (BBCH 69) and in August at harvest (BBCH 97). The actual measurement dates are presented in Table 2. Soil was sampled down to 2.3 m depth using a piston auger with an inner diameter of 0.025 m. Six subsamples were taken in each plot. The subsamples were divided into depth intervals of 0–0.5 m, 0.5–1.0 m, 1.0–1.5 m, 1.5–2.0 m and 2.0–2.3 m. The six replicate subsamples were combined into one bulk sample for each soil layer. The soil samples were frozen until being thawed in a refrigerator overnight before analysis. From each sample 20 g fresh weight soil were extracted in 100 mL 1 M KCl for 45 min. The soil extract was filtered and analysed for nitrate N and ammonium N by standard colorimetric methods using an AutoAnalyzer 3 (Bran+Luebbe). Only the nitrate N is shown in the results, because nitrate is the leachable form of N. The ammonium N level was low, and especially in deeper soil layers it was often below the detection level. Soil water contents were determined from wet weights subtracted dry weights measured after drying at 80 ◦ C for 48 h. The analytical values were converted into kg nitrate N ha−1 using the following average bulk density values for the different soil layers: 1515 kg m−3 for 0–0.5 m, 1615 kg m−3 for 0.5–1.0 m and 1620 kg m−3 for 1.0–2.3 m.

I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

41

Table 2 Overview of treatments and operations during the experiment. Operations marked with the same letter (a–g) were conducted within the same period. Treatment

Exp. 1 2011–12

Exp. 2 2012–13

Previous crop Ploughing Winter wheat cultivars

Oat 22.08.11 Hereford Cordiale Genius JB Asano 28.09.11 320 23.03.12 01.05.12 23.03.12 14.10.11 and 18.05.12 30.05.12

Winter barley 06.08.12 Hereford Cordiale Tabasco Tuareg 20.09.12 300 18.04.13 17.05.13 18.04.13 20.09.12 and 22.10.12 30.05.13 and 21.06.13 21.06.13 07.11.12 07.12.12 08.03.13 03.05.13 12.06.13 25.07.13 15.11.12 18.03.13 08.05.13 13.06.13 06.08.13 24.08.12 13.11.12 10.04.13 19.06.13 28.08.13

Winter wheat sowing date Seeding density (plants m−2 ) Fertilizer 20, 85, 150 kg N ha−1 Fertilizer +100/200 up to 250/350 kg N ha−1 Fertilizer 18/33a kg P ha−1 and 63 kg K ha−1 Herbicides Fungicides Insecticides Root measurements

Biomass sampling

Soil sampling

a b

b c d e f g b d e f g a b d f g

01.11.11 07.12.11 05.03.12 15.04.12 10.06.12 26.07.12 20.11.11 21.03.12 18.04.12 19.06.12 09.08.12 24.08.11 23.11.11 20.03.12 30.06.12b 06.08.12

18 kg P ha−1 in 2012 and 33 kg P ha−1 in 2013. Only in Genius and Hereford.

2.4. Definition of N efficiency

3.2. Effect of N fertilization on root growth

There exist several definitions and evaluation methods for nitrogen use and uptake efficiency (Good et al., 2004). In this study, nitrogen use efficiency (NUE) is defined as the extra grain yield harvested for each increase in applied N. Nitrogen uptake efficiency (NUpE) is defined as the percentage of the extra applied N that was harvested in plant.

The N effect on root growth was evaluated from means across cultivars, despite different set of cultivars for the two years, as statistical analyses showed no interaction effect of cultivar and N fertilization. The total root growth, measured as root intensity, was affected by N fertilization. However, the effect on root intensity differed between the two years. The high N fertilization levels increased root intensity in exp. 1, whereas an optimal N fertilization rate of 85–150 kg N ha−1 , with reduced root growth at higher or lower rates, was indicated in exp. 2 (Figs. 2 and 3). These effects were visible in soil layers below 0.5 m depth. In July, the total root intensities across cultivars in exp. 1 were 1.3 times higher in 250 N compared to 85 N treatments, whereas in exp. 2, the 250 N treatments resulted in 1.4 times less total root intensity (Fig. 2). To some extent, the root growth tendencies observed after fertilization were present already before fertilization (data not shown). Thus, the N effects on root growth should be interpreted with some caution, as initial field variability may have caused part of the observed effects. Especially the deviating and low root intensity in the upper 1 m soil layer of Hereford 250 N in exp. 1 (Fig. 3a) was present even before the N application. N fertilization did not affect root depth significantly, as no significant differences in root depth penetration rate (RDPR) were observed between the 85 N and 250 N treatments. Overall, higher root depth and root intensity were observed during exp. 2 (Figs. 4 and 5). The equation for RDPR was y = 0.7x − 87.1 (R2 = 0.78), and y = 1.0x − 69.9 (R2 = 0.89) in exp. 1 and 2, respectively. According to this, the average RDPR was 0.7 and 1.0 mm ◦ C day−1 in exp. 1 and 2, respectively. The average maximum root depth was 1.1 m and 1.5 m in exp. 1 and 2, respectively, when calculated as the average of measured root depths in June and July across cultivars and N fertilizer levels. Root intensities were high and roughly equal in the soil layers above 1 m, and declining below

2.5. Statistical analyses Significant differences in root growth, in aboveground biomass and N, in yield and in soil nitrate N were tested using general linear models (SAS GLM procedure) and pairwise t tests (Proc GLM, SAS Institute Inc., Cary, NC, USA). In cases where overlap of standard errors (SE) for the different treatments would make the figure information difficult to read, the least significant differences (LSD) were provided instead. In assessing differences between results, tests with P < 0.05 were considered statistically significant.

3. Results 3.1. Weather and growth conditions The weather conditions during the two experiments are shown in Fig. 1. In July and August 2011 precipitation was almost twice as high as average, which apparently caused a major loss of soil nitrate N through leaching or denitrification in the beginning of exp. 1. Another important deviation from a normal Danish weather pattern was the low temperatures in March and April 2013, which caused a delayed spring growth of the wheat in exp. 2.

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I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

Table 3 Soil nitrate N (kg ha−1 ) measured in different winter wheat cultivars fertilized with 85 and 250 kg N ha−1 . The table shows soil nitrate N measured in August before sowing, in November before winter, in March/April in early spring before fertilization, in June at anthesis and in August at harvest, summarized in 0–1 m and 1–2.3 m soil layers. Standard errors are presented in brackets, n = 3. Values within year and soil layer followed by different letters are statistically different (P < 0.05). Depth (m)

Exp. 1 2011–12

0–1.0 1.0–2.3

August before sowing 31.1 (7.2) 25.8 (5.0) Cordiale Hereford November 20.5 (0.2) 32.6 (9.5) 15.1 (2.8) 13.1 (1.7) March 29.3 (11.7) 22.5 (3.7) 23.6 (5.7) 17.1 (2.9) June 85 N 85 N 10.7 (0.7) 15.4 (3.5) August 16.2 (2.7) 11.4 (2.0) 16.1 (4.2) 17.9 (3.0)

Depth (m)

Exp. 2 2012–13

0–1.0 1.0–2.0*

0–1.0 1.0–2.0* 0–1.0 1.0–2.3

0–1.0 1.0–2.3

0–1.0 1.0–2.3

0–1.0 1.0–2.3 0–1.0 1.0–2.3

0–1.0 1.0–2.3 0–1.0 1.0–2.3 *

August before sowing 55.7 (3.5) 37.0 (4.8) Cordiale Hereford November 64.1 (8.0) 61.5 (5.0) 29.6 (3.1) 27.5 (0.4) April 44.2 (5.7) 38.1 (2.2) 49.9 (4.5) 41.4 (3.8) June 85 N 85 N 10.6 (0.7) a 11.3 (2.0) a 34.5 (3.7) 34.4 (11.5) August 21.1 (4.6) a 31.7 (5.5) b 34.6 (9.5) abc 24.3 (4.1) a

Genius

JB Asano

Hereford

Cordiale

Genius

JB Asano

24.9 (7.2) 14.4 (3.3)

27.6 (5.7) 12.2 (2.2)

22.2 (5.1) 22.8 (7.1)

24.5 (3.4) 16.6 (1.5)

85 N 10.2 (2.3) 20.1 (3.2)

85 N

250 N 33.6 (13.8) 18.4 (6.7)

250 N

250 N 39.9 (11.9) 15.8 (1.6)

250 N

13.3 (2.8) 20.1 (5.7)

14.3 (3.1) 12.8 (1.5)

39.9 (13.2) 19.9 (4.6)

21.0 (2.1) 15.0 (1.4)

22.1 (4.5) 18.1 (2.8)

32.7 (17.2) 20.9 (5.2)

Tabasco

Tuareg

Hereford

Cordiale

Tabasco

Tuareg

69.6 (7.2) 32.2 (8.3)

57.1 (4.2) 31.5 (3.7)

38.6 (3.0) 44.6 (1.8)

36.9 (4.0) 43.0 (1.8)

85 N 13.2 (2.4) a 35.8 (6.8)

85 N 11.1 (0.8) a 35.0 (7.4)

250 N 30.2 (6.8) b 34.5 (1.4)

250 N 36.7 (0.6) b 45.6 (3.9)

250 N 27.7 (2.3) b 38.7 (1.8)

250 N 37.2 (8.3) b 40.9 (6.6)

23.5 (2.2) ab 32.0 (1.2) abc

27.4 (1.7) ab 25.7 (5.3) ab

51.0 (1.1) cd 36.7 (5.4) bcd

64.4 (8.0) d 49.8 (8.1) de

46.0 (6.2) c 55.5 (8.7) e

50.5 (5.7) cd 42.3 (3.2) cde

In August and November 2011 the deepest measurements were at 2.0 m.

this depth. Below 1.5 m, root intensities were low, and in exp. 1, almost no roots were seen below 1.5 m. 3.3. Cultivar differences in root growth By the end of autumn the average root depth was 0.2 m and 0.5 m in exp. 1 and 2, respectively, with no significant depth differences among cultivars (Fig. 4). All cultivars showed active root growth during the winter period from December

Soil layer (m)

0.0

to March. On average, the cultivars had grown approximately 0.3–0.4 m deeper during both winters (Fig. 4), and total root intensity increased 4.3 and 1.4 times in exp. 1 and 2, respectively (Fig. 5). In general, all cultivars showed a strong root growth from late spring in April/May until anthesis in June. In this short period, root depth increased by approximately 0.4–0.5 m in both years (Fig. 4) and total root intensity increased 3.4 and 2.6 times in exp. 1 and 2, respectively (Fig. 5). After anthesis, further root growth was limited

a Exp. 2

Exp. 1

b

0.5 1.0

*

1.5

85 N 250 N

85 N 250 N

2.0 2.5 0

10

20

30

40

50

0

10

20

30

40

50

Root intensity (intersections/m gridline) Fig. 2. Effect of low and high N fertilization levels on winter wheat root growth. Depth distribution of root intensity determined across winter wheat cultivars at the end of the growing season. (a) Exp. 1 (2011–12) average of the cultivars Hereford, Cordiale, Genius and JB Asano. (b) Exp. 2 (2012–13) average of the cultivars Hereford, Cordiale, Tabasco and Tuareg. The figure shows the effect of 85 and 250 kg N ha−1 , respectively. Error bars represent standard error, n = 12. Statistically different values are indicated with *(P < 0.05).

I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

Soil layer (m)

0.0

43

a Exp. 2

Exp. 1

b

0.5

*

1.0

*

20 N

1.5

20 N

85 N

85 N

2.0

150 N 250 N

2.5

350 N

150 N 250 N 350 N

0

10

20

30

40

50

60

70 0

10

20

30

40

50

60

70

Root intensity (intersections/m gridline) Fig. 3. Effect of five N fertilization levels on winter wheat root growth. Depth distribution of root intensity determined on the winter wheat cultivar Hereford at the end of the growing season. (a) Exp. 1 (2011–12). (b) Exp. 2 (2012–13). The figure shows the effect of 20, 85, 150, 250 and 350 kg N ha−1 . Bars represent least significant difference, n = 3. Statistically different values are indicated with *(P < 0.05).

and to some extent root decay was even observed in the upper soil layers. Significant cultivar differences in average maximum root depth were only observed in exp. 2 in June, where Hereford reached a depth of 1.7 m, which was 0.3 m deeper than the other cultivars (Fig. 4). Though not significantly, Hereford also showed the deepest root growth for the rest of the season. In the same experiment, Hereford had the highest root intensity down the profile already from March and onwards (Fig. 5). Hereford was also among the deepest rooted cultivars in exp. 1 and it had the highest intensity from anthesis and onwards. Although differences between cultivars were in general small, Hereford had a tendency to higher RDPR than the other cultivars, especially at the low N fertilizer level. RDPR in the 85 N treatments were 0.1–0.4 mm ◦ C day−1 higher in Hereford than in the other cultivars (data not shown). The cultivar Genius had a tendency toward less root depth as well as less intensity until late spring after which it caught up with the other cultivars (Figs. 4 and 5). 3.4. Effect of N fertilization on soil N depletion, yield and N uptake

0.0

Exp. 1

Exp. 2

0.5

1.0

2011

2012

Sep

J an F eb Mar

Sep Oc t Nov Dec

Apr May J un J ul Aug

Sep Oc t Nov Dec

2.0

Apr May J un J ul Aug

**

Hereford Cordiale Genius J B As ano T abas c o T uareg

1.5

J an F eb Mar

Average max root depth (m)

In general, the amounts of soil nitrate N in the experimental fields were low, but it was substantially lower in exp. 1 than in exp. 2 (Table 3). This was seen as lower soil N content, lower N uptake in the wheat already in November and lower N uptake at harvest. At final harvest, the total N content in the crop fertilized with only

2013

Fig. 4. Average maximum root depth over time in the winter wheat cultivars Genius and JB Asano in exp. 1, Tabasco and Tuareg in exp. 2 and Hereford and Cordiale in both seasons. The figure shows the average effect of 85 and 250 kg N ha−1 . N was applied after the March measurements. Bars represent least significant difference. November and December: n = 3, March and after: n = 6. Statistically different values are indicated with **(P < 0.01).

20 kg N ha−1 was 54 kg N ha−1 in exp. 1, whereas it was 102 kg N ha−1 in exp. 2 (Table 4). Increasing the amount of N fertilizer in spring resulted in more nitrate left in the soil around anthesis in June and even at harvest in August. This effect was visible not only in the upper soil layers, but also in the subsoil (Table 3). The effect of N fertilization on soil N could be evaluated from means across cultivars, despite different set of cultivars for the two years, as statistical analyses showed no interaction effect of cultivar and N fertilization. Across cultivars, the 250 N treatments left significantly more N in the upper soil layer at harvest compared to the 85 N treatments (Fig. 6). In exp. 2, this effect was also highly significant below 1 m soil depth, as the high N treatment had left on average 17 kg nitrate N ha−1 more in the soil layers from 1 to 2.3 m depth. In these deep layers, the average N depletion from June until August was 6 kg nitrate N ha−1 in the 85 N treatments, whereas the soil N content had increased with an average of 6 kg nitrate N ha−1 in the 250 N treatments. Likewise, the comparison of a broader range of N fertilizer rates in a single cultivar showed that increasing the N fertilizer amount resulted in more nitrate left in upper as well as in deeper soil layers at harvest (Fig. 7). Soil nitrate N in layers below 0.5 m was almost depleted in 20 N, 85 N and 150 N treatments, but at higher N fertilization rates the soil nitrate N at harvest was increased, also in the deeper layers. When increasing the N fertilizer amount from 150 to 250 kg N ha−1 , up to 37% of the extra 100 kg N ha−1 was left in the soil at harvest. With a further increase from 250 to 350 kg N ha−1 , up to 90% of the extra N was left in the soil (Table 4). Aboveground measurements at anthesis revealed significant effects of N fertilization on biomass, N concentration and N content (Appendix Table A.1 of Supplementary material). In general, the biomass at anthesis was lower in exp. 2. In both years, the 250 N treatment had the highest biomass, slightly higher than the 350 N treatment, which showed the highest N concentration and the highest plant N content. Grain yields were highest at 250 N in exp. 1 and at 150 N in exp. 2, and likewise maximum harvest index of 56 and 55, respectively, were measured at these N treatments (Table 5). No significant difference in grain yield was observed between 150 N and 350 N treatments, but in exp. 1, there was a significant yield increase from 150 N to 250 N treatment. The N concentration was highest at the 350 N treatment, resulting in the highest grain N content in this treatment despite the lower grain yield. The N harvest index decreased with increased N fertilization, as an increased percentage of the total N was embedded in straw. Increasing N fertilization from 150 to 250 kg N ha−1

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I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

0.0

a

Exp. 1

b

Exp. 2

*

0.5 1.0 1.5 2.0 2.5 0.0

01.11.2011 c

07.11.2012 d

07.12.2011

07.12.2012

e

f

0.5 1.0 1.5 2.0 2.5 0.0 0.5

**

1.0

Soil layer (m)

1.5 2.0

05.03.2012

2.5 0.0

g

08.03.2013 h

15.04.2012

03.05.2013

i

j

10.06.2012

12.06.2013

k

l

0.5

*

1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0

2.0

Hereford Cordiale Genius JB Asano

2.5

26.07.2012

1.5

0

10

20

30

40

50

Hereford Cordiale Tabasco Tuareg

25.07.2013 0

10

20

30

40

50

Root intensity (intersections/m gridline) Fig. 5. Depth distribution of winter wheat root intensities during the season. (a, c, e, g, i, k) Exp. 1 (2011–12) root intensities of the cultivars Hereford, Cordiale, Genius and JB Asano. (b, d, f, h, j, l) Exp. 2 (2012–13) root intensities of the cultivars Hereford, Cordiale, Tabasco and Tuareg. After the March measurements, all cultivars were applied 85 and 150 kg N ha−1 . Prior to the June measurements further 100 kg N ha−1 were applied up to 250 kg N ha−1 . The figure shows the average root intensities of both N treatments. The dates of measurements are indicated in the graphs. Bars represent least significant difference. November and December: n = 3, March and after: n = 6. Statistically different values are indicated with *(P < 0.05) and **(P < 0.01).

I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

45

Table 4 Applied N fertilizer in spring compared to the amount of soil nitrate N and total plant N measured at harvest in winter wheat (cv Hereford). The percentage of the extra applied N that was left in the soil or harvested in plant (nitrogen uptake efficiency, NUpE) is indicated for each increase in applied N. The amount of extra grain yield harvested for each increase of applied N is defined as nitrogen use efficiency (NUE). Year

Exp. 1 2011–12

Exp. 2 2012–13

Applied N in spring (kg ha−1 )

Soil nitrate N at harvest (kg ha−1 )

Percentage of extra applied N left in soil at harvest (%)

Plant N at harvest

NUpE

NUE

(kg ha−1 )

(%)

(kg kg−1 )

20 85 150 250 350

31 28 23 60 150

0 0 37 90

54 91 134 251 282

56 67 117 31

45 33 15 −3

20 85 150 250 350

52 45 53 88 173

0 12 35 85

102 143 199 229 276

64 86 30 47

37 21 −4 1

resulted in a plant N increase of 117 and 30 kg ha-1 , corresponding to a NUpE of 117 and 30% in exp. 1 and 2, respectively (Table 4).

effect was most evident in exp. 1, where Hereford compared to the average of the other cultivars, had an increased grain yield of 1.2 and 1.3 Mg ha−1 in the 85 N and 250 N treatments, respectively (Table 5). Although only significant in exp.1, the N concentration was in general lower in Hereford at both N fertilizer levels. Consequently, in most cases the lower N concentration resulted in slightly lower total N content ha−1 in Hereford, despite a higher grain yield. Only in exp. 2 in the 85 N treatments, the N content ha−1 was actually higher in Hereford than in the other cultivars.

3.5. Cultivar differences in soil N depletion, yield and N uptake Soil nitrate measurements in autumn and early spring before fertilization did not reveal any significant cultivar differences in soil nitrate N (Table 3). However, at harvest in exp. 2, there was a tendency that Hereford had depleted soil nitrate N in the deep layers more than the other cultivars, especially in the high N treatment (Table 3). Aboveground measurements of biomass, N concentration and N content during the season up to and including anthesis did generally not reveal clear patterns of cultivar differences (Appendix Table A.1 of Supplementary material). At harvest, Hereford had the highest grain yield and consequently higher NUE compared to the other cultivars, as it produced more grain per N supply. This 0.0

4.1. General results on wheat root growth Crop rooting depth and hence the potential zone of water and nutrient uptake can be calculated by means of estimated root

a Exp. 2

Exp. 1

b

**

0.5

*** ***

*

1.0

85 N 250 N

***

85 N 250 N

1.5

Soil layer (m)

4. Discussion

2.0

June 2.5 0.0

June d

c

**

0.5

*** *** ***

1.0 1.5

*

2.0

August

August

2.5 0

10

20

30

40

0

10

20

30

40

-1

Nitrate N (kg ha ) Fig. 6. Effect of low and high N fertilization levels on nitrate N content down the soil profile across winter wheat cultivars. (a, c) Exp. 1 (2011–12) average of the cultivars Hereford, Cordiale, Genius and JB Asano. (b, d) Exp. 2 (2012–13) average of the cultivars Hereford, Cordiale, Tabasco and Tuareg. (a, b) Measurements conducted in June after fertilization. (c, d) Measurements conducted in August around crop harvest. The figure shows the average soil nitrate N content under all cultivars applied 85 and 250 kg N ha−1 , respectively. Error bars represent standard error, n = 12. In June 2012, soil N was only measured in Hereford and Genius, n = 6. Statistically different values are indicated with *(P < 0.05), **(P < 0.01) and ***(P < 0.001).

46

I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

Soil layer (m)

0.0

Exp. 1

a Exp. 2

b

***

*

0.5

***

1.0 1.5 2.0 2.5 0

20

*

Mar. bef ore N 20 N 85 N 150 N 250 N 350 N 40

100

Apr. bef ore N 20 N 85 N 150 N 250 N 350 N

*

0

20

40

100

-1

Nitrate N (kg ha ) Fig. 7. Effect of five N fertilization levels on nitrate N content down the soil profile in the winter wheat cultivar Hereford in August around crop harvest. (a) Exp. 1 (2011–12). (b) Exp. 2 (2012–13). The figure shows the effects of 20, 85, 150, 250 and 350 kg N fertilizer ha−1 . Soil nitrate in March/April before fertilization is indicated with the gray line. Bars represent least significant difference, n = 3. Statistically different values are indicated with *(P < 0.05) and ***(P < 0.001).

depth penetration rates (RDPR). The estimated RDPR of 0.7–1.0 mm ◦ C day−1 in the present experiments were lower than the winter wheat root growth rate of 1.3 mm ◦ C day−1 found using a similar method in a comparable sandy loam Agrudalf soil (ThorupKristensen et al., 2009). Differences in RDPR even on similar soil types are not unusual, as they reflect variations in the soil characteristics at the sites (Kirkegaard and Lilley, 2007). Until fertilization of the present experiments, the amounts of inorganic N in the topsoil as well as in the subsoil were substantially lower compared to the study by Thorup-Kristensen et al. (2009), which could cause the lower RDPR (Gao et al., 1998; Kristensen and ThorupKristensen, 2007). An interesting observation was that all cultivars had active root growth during the winter period. Winter root growth may be important for the main season growth, as it improves the root contact with resources by the onset of the main growing period.

The strong root growth found in all cultivars in the period during stem elongation until anthesis followed by only limited root growth until harvest corresponded to findings in other field studies of winter wheat (Gregory et al., 1978; Barraclough and Leigh, 1984; Kuhlmann et al., 1989; Kirkegaard and Lilley, 2007). A generally lower root density and depth was observed in exp. 1 compared to exp. 2, which could be the effect of the unusually high precipitation in the summer prior to exp. 1. The high rain fall caused a low soil N content, most likely due to increased N leaching loss or denitrification. In November, the measured nitrate N in the 0–2 m soil profile was on average 40 kg N ha−1 . This value was only half of the average 80 kg N ha−1 found in exp. 2 in November, which is a much more typical value for this soil type, vegetation and time of year according to simulation studies (Pedersen et al., 2009). The low initial soil N content in exp. 1 may have caused reduced root growth. The wet soil in itself may also have reduced the root growth, if low

Table 5 Harvest yield of winter wheat cultivars at different N fertilization rates. The table shows dry matter of grain (DM grain), dry matter of straw (DM straw), harvest index (HI), N content in grain (N grain), N content in straw (N straw), N harvest index (NHI), N concentration in grain (N% grain) and N concentration in straw (N% straw). Standard errors are presented in brackets, n = 3. Values within year and measurement followed by different letters are statistically different (P < 0.001). Cultivar

Applied N −1

(kg ha

)

DM grain −1

(Mg ha

DM straw

)

Exp. 1 2012 Hereford Hereford Cordiale Genius JB Asano Hereford Hereford Cordiale Genius JB Asano Hereford

20 85 85 85 85 150 250 250 250 250 350

4.2 (0.5) a 7.1 (0.3) c 5.7 (0.2) b 5.6 (0.4) b 6.3 (0.4) bc 9.2 (0.5) de 10.7 (0.2) f 9.8 (0.2) def 9.4 (0.3) de 9.1 (0.9) d 10.4 (0.2) ef

Exp. 2 2013 Hereford Hereford Cordiale Tabasco Tuareg Hereford Hereford Cordiale Tabasco Tuareg Hereford

20 85 85 85 85 150 250 250 250 250 350

6.3 (0.6) a 8.6 (0.2) bcd 7.7 (0.4) b 8.1 (0.3) bc 7.8 (0.2) b 10.0 (0.2) e 9.6 (0.3) de 9.1 (0.3) cde 9.1 (0.4) cde 9.0 (0.2) cde 9.7 (0.4) e

HI

N grain −1

N straw )

NHI

N grain

(%)

(%)

N straw

(%)

(kg ha

3.7 (0.3) a 6.3 (0.3) c 5.3 (0.2) b 5.6 (0.2) bc 6.3 (0.4) c 7.6 (0.4) d 8.3 (0.5) de 7.7 (0.2) d 7.9 (0.2) de 8.7 (0.4) e 8.5 (0.1) de

53 53 52 50 50 55 56 56 54 51 55

46 (4) a 75 (5) ab 82 (10) b 77 (6) ab 80 (8) b 107 (6) b 189 (9) c 209 (10) c 195 (15) c 189 (23) c 209 (11) c

8 (1) a 15 (1) ab 15 (1) ab 15 (1) ab 17 (2) ab 27 (2) b 62 (11) de 49 (6) cd 48 (8) c 64 (2) e 73 (4) e

86 83 84 84 82 80 75 81 80 75 74

1.1 (0.03) a 1.1 (0.03) a 1.4 (0.16) c 1.4 (0.02) bc 1.3 (0.04) abc 1.2 (0.04) ab 1.8 (0.05) d 2.1 (0.11) e 2.1 (0.13) e 2.1 (0.08) e 2.0 (0.08) de

0.2 (0.01) a 0.2 (0.01) ab 0.3 (0.02) ab 0.3 (0.01) ab 0.3 (0.01) ab 0.4 (0.01) b 0.7 (0.09) cde 0.6 (0.06) cd 0.6 (0.09) c 0.7 (0.03) de 0.9 (0.04) e

5.2 (0.5) a 7.0 (0.2) bc 6.4 (0.3) b 7.1 (0.1) bc 6.7 (0.2) b 8.2 (0.1) de 8.2 (0.2) de 7.6 (0.3) cd 8.2 (0.2) de 8.0 (0.1) de 8.8 (0.3) e

55 55 55 53 54 55 54 55 53 53 52

87 (4) a 119 (7) b 112 (8) b 112 (8) b 114 (2) b 160 (6) c 171 (2) cd 182 (4) d 172 (2) cd 182 (4) d 200 (7) e

15 (2) a 24 (2) b 21 (2) ab 23 (1) b 22 (2) b 40 (2) c 58 (3) e 49 (3) d 54 (3) de 50 (1) d 75 (3) f

85 83 84 83 84 80 75 79 76 78 73

1.4 (0.09) a 1.4 (0.07) a 1.5 (0.03) ab 1.4 (0.05) a 1.5 (0.02) ab 1.6 (0.04) b 1.8 (0.04) c 2.0 (0.05) de 1.9 (0.07) cd 2.0 (0.03) de 2.1 (0.02) e

0.3 (0.01) a 0.3 (0.02) a 0.3 (0.02) a 0.3 (0.01) a 0.3 (0.02) a 0.5 (0.02) b 0.7 (0.03) d 0.7 (0.02) cd 0.7 (0.02) cd 0.6 (0.02) c 0.9 (0.02) e

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oxygen conditions in the soil inhibited germination and early root development. In addition, the wet soil conditions caused problems doing the root observations, as mud covered parts of the minirhizotrons when installed. This condition reduced the number of roots, which could be observed through the mud-covered glass and might have influenced the observed root intensities negatively. Though, as the problem with mud existed in the entire field, it did not influence the comparative studies of cultivar and N treatment effect on root growth. Root depth and intensity was measured using the minirhizotron technique, which allows repeated non-destructive observations. This technique is widely used and evaluated, and it is one of the best tools available for directly studying roots (Hendrick and Pregitzer, 1996; Johnson et al., 2001). The method has some risk of artefacts, as roots may be affected by growing onto the tube surface, as well as by the soil structural effects caused by the drilling needed for installing the minirhizotrons. Problems with preferential root elongation along the minirhizotron are reduced by installing the tubes at an angle from vertical, typically 30◦ or 45◦ (Johnson et al., 2001), and only recording the roots on the upwards facing surface. Previous studies have shown good relationships between minirhizotron root measurements and uptake of soil nitrogen (Thorup-Kristensen, 2001; Kristensen and ThorupKristensen, 2004), making the method well suited for the purpose of this study. 4.2. Effect of N fertilization on root growth N fertilization clearly affected root growth. The effects were observed mainly as differences in root density in soil layers below 0.5 m, where root density was increased when increasing N fertilization from 20 to 150 kg N ha−1 . The effect of further N fertilization varied between the two experiments. The results did not reveal effects of N fertilization on root depth, indicating that N fertilization affected root branching more than it affected root depth. Significant effects of N treatments on root density without effects on root depth have also been indicated in other field studies (Thorup-Kristensen and van den Boogaard, 1999). In exp. 1, root density continued to increase with increasing N fertilization, while in exp. 2, the optimum was at 150 kg N ha−1 , with decreasing root density at higher N fertilization. Although root densities are known to increase at higher soil N levels (Barraclough et al., 1989), there might be an optimal N level for root proliferation, above which root growth is not further increased or even is reduced, as indicated in other field studies (ThorupKristensen and van den Boogaard, 1999; Svoboda and Haberle, 2006). Considering the higher background N availability and lower general N response in exp. 2, the results do indicate that an optimum N level was reached here, whereas it was not reached in exp. 1. Though, it is important to keep in mind that root growth is affected by many factors, which could have differed between the two years, such as water stress. It is interesting that the most direct effects of N supply were observed in soil layers below 0.5 m, as this depth was well below the actually fertilized soil layer. In this part of the soil, roots were mainly formed after the time of fertilizer application. The effects of N fertilization on root growth may be due to direct effects on root branching. Though, as N fertilization affects tillering, it also affects root growth indirectly, as each tiller has nodal roots (Dai et al., 2014). N fertilization increases the number of well-developed tillers. However, high fertilization may cause excessive formation of tillers, of which many do not develop well and consequently, root growth will not be further increased. Incidental co-variability, especially in exp. 1, in root measurements made in spring before the N applications and the subsequent measured N effects indicate that the N effects must be interpreted

47

with some caution. However, most of the N effects on root growth were observed in deeper soil layers in which no or few roots were present in the measurements made before N fertilization. Extracting root intensity values measured before fertilization from values measured after fertilization did not change the root intensity values in the deep soil layers. 4.3. Cultivar root growth, soil N depletion, N uptake and yield Cultivar differences in root growth were seen, and the main result was that the cultivar Hereford showed more and deeper root growth, although only significantly at a few measurements. As the studied cultivars are all modern and high yielding it is reasonable that they do not show strong differences in root growth. Minor differences in root growth can be hard to detect under field conditions because several factors, such as soil structure, nutrients and water might influence the root growth and blur potential cultivar differences (Dracup et al., 1992; Tennant and Hall, 2001; Kirkegaard and Lilley, 2007). The trend of stronger root growth in the cultivar Hereford was mainly observed by the end of the growth season, and in exp. 2, it correlated with the tendency of reduced subsoil N content under this cultivar. Changes in subsoil N was assumed to be caused by plant N uptake from these layers, because leaching at this time of the season as well as mineralization in the subsoil were regarded negligible (Kuhlmann et al., 1989). However, the differences in root growth and subsoil N depletion among the cultivars were not reflected in the aboveground measurements of plant N, as in most cases Hereford did not show higher N uptake than the other cultivars. This could be due to the fact that measuring crop N content at harvest does not give a measure of the total crop N uptake during the growth season. During growth, N is also embedded in roots, root exudates and lost biomass. Since Hereford had a tendency to more roots, more N might be deposited in the soil as organic N. Even though the root growth differences among the cultivars did not seem strong, they could be important. In both years, the root density of Hereford was twice as high compared to the other cultivars in deep soil layers of 1.0–1.5 m in exp. 1 and 1.5–2.0 m in exp. 2, respectively. Deeper rooting is essential for the ability to use available resources like N and water from deeper soil layers (Thorup-Kristensen and Sørensen, 1999; Tennant and Hall, 2001; King et al., 2003). The slightly deeper and denser rooting of Hereford could be of importance during the grain filling stage. At this time, water resources often become scarce but have a more direct effect on grain production than water at an earlier stage (Manschadi et al., 2006; Kirkegaard et al., 2007). Therefore, the stronger root growth of Hereford might contribute significantly to its higher grain yield and NUE. That Hereford overall had a higher grain yield and a lower N concentration was predictable, since this cultivar is classified as high yielding fodder wheat, but part of the reason for this higher yield may be its stronger root growth. 4.4. Effect of N fertilization on yield and soil N depletion The wheat yields were generally high, as expected under the present growth conditions. Grain yields increased with N fertilization and levelled out at the high N fertilizer applications. Consistent with the higher initial soil N in exp. 2, the grain yields peaked at a lower N application in this experiment. Also as expected, the grain yield from extra applied N (NUE) decreased with increased N fertilizer level, especially at the high N treatments where NUE even turned negative. At the low N fertilization levels a relatively small part of the applied N, around 60%, was retrieved in plant and soil at harvest. This was expected, as part of the N fertilizer would be embedded

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I.S. Rasmussen et al. / Europ. J. Agronomy 68 (2015) 38–49

in roots, root exudates and soil microorganisms (Bradbury et al., 1993). On the other hand, at higher fertilization levels, a considerable part of the applied N amount, in more cases even more than 100%, was retrieved in plant and soil at harvest. It is not clear why such a high recovery was found when combining plant N and soil inorganic N, but increased N mineralization stimulated by high inorganic N levels may have contributed to this (Broadbent 1965; Ellum et al., 2013). Increasing N fertilization from 20 to 85 and 150 kg N ha−1 did not lead to an increase of inorganic N residues left in the soil after harvest. So, as long as the N supply stayed below the N uptake capacity of the wheat, increased N fertilization did not lead to increased amounts of soil nitrate at harvest. On the other hand, at the high fertilization levels of 250 and 350 kg N ha−1 , an increased amount of soil nitrate was found at harvest. This is important, as many North European wheat crops are fertilized with between 150 and 250 kg N ha−1 , and some even above 250 kg N ha−1 . Under the stricter Danish nitrogen regulations (Dalgaard et al., 2014) wheat crop fertilization is around 150 kg N ha−1 . The present study indicates that under the given field and weather conditions, a spring application up to at least 150 kg N ha−1 did not increase soil N at harvest. N uptake efficiency is dependent not only on the root system but also on plant demand. Therefore, the N amounts left in the high N treatments reflected that crop demand became saturated as N fertilization increased to these rates, and not that the root system was insufficient for depleting the soil (Robinson et al., 1994; Thorup-Kristensen and Sørensen, 1999). Still, in the present study there was an N interval, especially between 150 and 250 kg N ha−1 , where more N was left in the soil, but at the same time more N was taken up. Within this range of N supply there could be a potential for future improvements, due to improved root growth, improved root N uptake kinetics or improved shoot sink strength for N. At the high N fertilization treatments, increased N amounts were left in the entire soil profile. The effect was most profound in the upper soil layer, but it was also significant in the subsoil. Even though the amount of increased N in the subsoil was less than in the upper soil layer it is not less important, as subsoil N is highly prone to leaching loss (Thorup-Kristensen and Nielsen, 1998). Especially in fields with higher initial subsoil N conditions than found in the present experiment, high fertilization rates could lead to a considerable loss of the existing N resources from the subsoil.

5. Conclusions • Winter wheat root density was increased by N fertilization up to 150 kg N ha−1 , above this level the effect varied between the two years. The most direct effects of N supply on root density were observed in soil layers below 0.5 m. N fertilization did not appear to affect root depth. • There were root growth differences among the winter wheat cultivars, as the cultivar Hereford showed tendencies to more and deeper root growth, and at least in one year this was correlated with a stronger subsoil N depletion. • NUpE differed strongly with N fertilizer level. At low and moderate N fertilization up to 150 kg N ha−1 , NUpE was high and fertilization did not increase the amount of nitrate left in the soil at harvest. NUpE decreased when fertilization was increased from 150 to 250 kg N ha−1 , and above 250 kg N ha−1 almost all the extra applied N was left in the soil at harvest. • The soil nitrate increase caused by high N fertilization was most profound in the upper soil layer, but it was also significant in the subsoil.

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