Interannual variability in root growth of spring wheat (Triticum aestivum L.) at low and high nitrogen supply

Europ. J. Agronomy 26 (2007) 317–326

Interannual variability in root growth of spring wheat (Triticum aestivum L.) at low and high nitrogen supply Juan M. Herrera 1 , Peter Stamp 2 , Markus Liedgens ∗ Institute of Plant Sciences, Swiss Federal Institute of Technology, Z¨urich, Switzerland Received 21 November 2005; received in revised form 21 September 2006; accepted 16 November 2006

Abstract Little is known about the spatial and temporal characteristics of the root growth of spring wheat and its modification by nitrogen (N) supply. For 4 years the cultivar Toronit was fertilized with 20 (LN) or 270 kg N ha−1 (HN) in lysimeters. The shoot traits were measured at harvest, while root growth was screened regularly at 10 soil depths in minirhizotrons between 0.05 and 1.00 m. The cumulative number of roots cm−2 (CNR) was fitted to a logistic equation to study the course of root growth at each soil depth. Furthermore, the vertical patterns of CNR were examined at the beginning of tillering, stem elongation, anthesis and physiological maturity by a non-parametric regression (splines). The parameters of the logistic and non-parametric models were influenced by all the factors; thus the root system was highly plastic. Whereas the N off take was similar at LN in 1999, 2001 and 2002, the period of linear increase in CNR in the subsoil was 7 d longer in 2001 than in 2002. At HN, the N off take was higher in 1999 than in 2001 but the reverse was true for root growth. There was also variation among years in the total duration of root growth, with differences up to 20 d. The percentage of roots grown after anthesis ranged from 1 to 22% of the total roots grown by physiological maturity, demonstrating that the root growth of spring wheat can be high and variable after anthesis. This percentage differed among years more in the subsoil and supported the evidence provided by the time parameters of the logistic equation that the impact of climatic and soil conditions on root growth seems to become stronger with time. At all levels of N supply, the vertical pattern of CNR was characterized by an exponential decrease at the beginning of tillering in all the years. Such a clear pattern was not found at later developmental stages. Though the basic knowledge of the variability of root growth of spring wheat increased, the interannual variability in root dynamics was not explained fully by climatic differences among the growing seasons. © 2006 Elsevier B.V. All rights reserved. Keywords: Wheat; Nitrogen; Root; Development; Growth; Soil

1. Introduction Root growth is a crucial process determining the impact of environmental effects on crop cultivation (Andr´en et al., 1993); it is thus a relevant factor, which must be taken into account with respect to agronomical practices.

Abbreviations: AN, anthesis; BT, beginning of tillering; CNR, cumulative number of roots cm−2 ; d, days; DAS, days after sowing; HN, high nitrogen supply (270 kg N ha−1 ); LN, low nitrogen supply (20 kg N ha−1 ); MNR, 50MNR, 50/75MNR, parameters of the logistic equation; NS, nitrogen supply; PM, physiological maturity; SE, stem elongation; SO, sowing; Y, year ∗ Corresponding author at: Eschikon 33, CH-8315 Lindau, Z¨ urich, Switzerland. Tel.: +41 52 354 9123; fax: +41 52 354 9119. E-mail address: [email protected] (M. Liedgens). 1 Present address: USDA-ARS, 2150 Center Avenue, Building D, Suite 100, Fort Collins, CO, USA. 2 Present address: Universit¨ atstrasse 2, CH-8092 Z¨urich, Switzerland. 1161-0301/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.eja.2006.11.003

Root growth over time has seldom been described. Gregory et al. (1978) observed an exponential increase in the root mass of winter wheat after sowing, followed by a linear increase up to anthesis. Barraclough and Leigh (1984) reported an exponential increase in root weight and length between sowing and anthesis. However, this pattern varied among the different environments. Campbell et al. (1977) showed that the root density increased exponentially in the top 0.90 m and linearly from 0.90 to 1.05 m up to the emergence of the flag leaf. The slopes of these curves were modified by N and soil moisture conditions and their interaction. Root growth can vary according to soil conditions, as water supply (Asseng et al., 1998) and water and N supply (Campbell et al., 1977). An important response to N is an increase in root branching (Belford et al., 1987), which occurs especially in soil patches, as shown by Drew and Saker (1975) for barley; this process is accompanied by an increase in the rate of N uptake (Moorby and Besford, 1983) and its translocation to the whole

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plant mass (Pan et al., 1985). N affects root length and, thus, the dynamics of the uptake of other elements such as P, Mg and Ca (Barraclough, 1986a). A greater N availability was reported to reduce root length density (Comfort et al., 1988) at deeper soil layers in relation to the topsoil. However, Belford et al. (1987) and Campbell et al. (1977) found no such effect. Furthermore, seminal roots tend to be relatively less responsive to changes in N availability than nodal roots (Wang and Below, 1992). Therefore, the effect of N availability on the root system changes throughout the growing season, and the spatial and temporal patterns of root growth will determine the ability of wheat to access the available N sources (Belford et al., 1987). Because of the mobility of N in the soil, high root densities may not represent a competitive advantage in the uptake of N available from the soil. However according to King et al. (2003), N uptake is probably responsive to root interception, early in the growing season or in deep soil layers later in the growing season. High nitrate uptake was reported until the end of grain filling (Page et al., 1978; Barraclough, 1986a). Root growth continues after anthesis (Gregory et al., 1978; Asseng et al., 1998); it is thus probably relevant for N uptake and must be properly quantified. A better understanding of root growth may be essential to achieving better synchronization between N fertilization and N uptake and, hence, to obtaining optimum N use efficiency in spring wheat. The objective of the present study was to quantify the variability in the temporal and vertical growth of the root system of spring wheat during four growing seasons. Effects of year and N supply on root growth were evaluated by frequently recording root growth at the soil–minirhizotron interface at 10 different soil depths. 2. Materials and methods 2.1. Experimental conditions The spring wheat (Triticum aestivum L.) cultivar, Toronit, grew for 4 years (1999–2002) in the Swiss midlands near Zurich (47◦ 26 N, 8◦ 40 E) in drainage lysimeters containing minirhizotrons to enable the observation of the roots. Each lysimeter unit was a watertight, double-walled container made of fiberglass. The inner surface area of the container was 1.00 m2 and the soil column 1.10 m deep. Each lysimeter held 10 minirhizotrons located at a depth between 0.05 and 1.00 m. The horizontally installed minirhizotrons were 1.20 m long with an external diameter of 60 mm. The lysimeters were artificially repacked with topsoil (0.00–0.30 m) taken from a nearby sandy loam soil, with the exception of the lower section of the column which consisted of two layers of glass foam and three layers of quartz sand. Soil filling was done in 1993 and then in 2000 to replace broken minirhizotrons, finishing just before the sowing of the crop in spring. Table 1 shows physical and chemical properties of the soil used to fill the lysimeters. A more detailed description and evaluation of the facility can be found in Liedgens et al. (2000). Spring wheat was sown on 15 March 1999, 23 March 2000, 3 April 2001 and 8 March 2002. The seeding rate was 420 seeds m−2 and the sowing depth 20–30 mm. The rows were

Table 1 Physical and chemical properties of the soil used to fill the lysimeters Parameter

Value

Year Sand (%) Loam (%) Clay (%) PH Organic matter (%, Blake-Walkley) Assimilable K2 O (ppm, NH4 acetate) P (g kg−1 soil, Olsen) Initial total N (kg N ha−1 , Kjeldahl)

1999

2000 54 29 17

7.2 3.0 36.5 0.4 24.5

8.0 2.8 26.7 0.4 21.0

0.14 m apart and were perpendicular to the minirhizotrons. Twenty days after the first sowing date in 1999, two lysimeters of eight were re-sown due to poor emergence. Each year, before sowing, fertilizer was applied at rates of 60 kg ha−1 of Foskal® (7, 20, 1, 4 and 2 kg ha−1 P, K, Mg, Ca and S, respectively) and 20 kg N ha−1 as ammonium nitrate (NH4 NO3 ). An additional 250 kg N ha−1 (HN), split into four applications of NH4 NO3 (90, 40, 60 and 60 kg N ha−1 ), were added to half the lysimeters, while the other lysimeters were not further fertilized (LN). The split N in the HN treatment was applied at 45, 64, 77 and 92 DAS in 1999, at 26, 55, 76 and 85 DAS in 2000, at 42, 67, 75 and 90 DAS in 2001 and at 46, 66, 72 and 110 DAS in 2002. The crop was sprayed with a growth regulator (Moddus® ) at a rate of 0.5 l ha−1 at the 1st node stage (BBCH 30–32). The lysimeters were irrigated (10 mm) during the early dry season in 1999, during grain filling in 2000 (35 mm) and between stem elongation and anthesis in 2002 (35 mm). For 10 d during grain filling in 2002, the experiment was covered with a net, which reduced incident solar radiation by approximately 12% but protected the experiment during a hail storm. The experimental layout in each year was a randomized complete block design with three (2001 and 2002) or four replications (1999 and 2000). 2.2. Data sampling and screening Meteorological data were obtained from the nearby weather station. The harvest took place 9 August 1999, 7 August 2000, 14 August 2001 and 29 July 2002. All the shoots per lysimeter were cut at ground level at physiological maturity (BBCH stage 92 or later), dried at 65 ◦ C for 48 h and separated into grains and straw. The N concentration of the straw and in the grains was determined with a LECO CHN-1000 auto analyzer (LECO Corporation, St Joseph, MI, USA) and enabled an estimation of the shoot N off take. Root images were taken in the minirhizotron from the upper surface of the minirhizotron–soil interface by means of a special camera system (Bartz Technology Co., Santa Barbara, CA, USA) from a strip 18 mm wide and 202.5 (1999) or 243 mm (2000, 2001 and 2002) long, corresponding to 15 and 18 single images, respectively. Each image had a size of 13.5 by 18.0 mm. Single images were converted into a digital format using a frame grabber. All the images recorded over the entire season at the same position in the minirhizotron were organized in an image

J.M. Herrera et al. / Europ. J. Agronomy 26 (2007) 317–326

time series. This allowed for the sequential screening of new roots as they appeared on each image. The screening of the image time series was done block wise by trained operators. The number of roots (Crocker et al., 2003) was determined according to Upchurch and Ritchie (1983). Since there are no objective visual criteria to determine whether a root is active or not (Smit et al., 2000), root growth was assessed according to Box and Ramseur (1993) concentrating on the identification of new roots rather than on root decay, which had been already characterized by Bohm (1978) and Asseng et al. (1998). The response variable was the cumulative number of roots (CNR), i.e. the formation of new roots in time between sowing and the observation date. The values for each position were converted to a surface unit (cm2 ) and averaged over all the positions in the same minirhizotron. 2.3. Data analysis To obtain the CNR of each lysimeter by soil depth combination, measurements were repeated over time. One approach to analyzing these data is to extract the parameters of a suitable parametric model (Meredith and Stehman, 1991) and analyze their variance. The raw data of CNR indicated that a logistic equation (Hunt, 1982) would be a good approximation of the pattern observed for CNR with time. The logistic model is characterized by three parameters: MNR, the asymptotic limit of CNR, 50MNR, the time at which 0.5 of the asymptotic limit is reached and 50/75MNR, the time lag between 50MNR and the time at which CNR reaches 0.75 of the asymptotic limit. A particular advantage of this modeling approach is that the three parameters can be interpreted biologically: MNR indicates the approximate magnitude of root growth, while 50MNR and 50/75MNR indicate the approximate time course of root growth. 50MNR is the time at which the inflexion point of the logistic equation determines the change from an exponential increase to a linear increase in CNR. The period of linear increase in CNR is indicated by 50/75MNR. The parameters of the logistic model were calculated for each lysimeter with the R function nlsList (Pinheiro and Bates, 2000). Besides CNR we also calculated the percentage of total roots at anthesis. The percentage of the total number of roots cm−2 grown at anthesis (PRA) is a measure of the relative period of root growth in the critical period for yield determination, defined as PRA = (CNRAN /CNRPM ) × 100. Finally, in order to study the vertical pattern of CNR along the soil profile at four growth stages, splines, a non-parametric regression, were fitted to the CNR data. The polynomial model

319

was parameterized starting from a full model and following a backward selection procedure (Crawley, 2002) and was evaluated according to Pinheiro and Bates (2000). The shoot data and the parameters of the logistic model were analyzed using the SAS® Proc Mixed (Littell et al., 1996). For each analysis, years and N supplies (NS) were set as fixed, while blocks were set as random factors, nested within years. Denominator degrees of freedom were calculated according to the Satterthwaite method, as implemented in SAS (Littell et al., 1996). The statistical analyses of the parameters of the logistic equation were performed separately for each soil depth. To account for heteroscedasticity, the statistical analysis of the parameters 50MNR and 50/75MNR of the logistic model and N concentrations of the straw and the grain were performed on log-transformed data. The non-parametric modeling of the CNR depth profiles was performed with the functions lme (Pinheiro and Bates, 2000), bs (Hastie, 1992) and smspline (Pinheiro and Bates, 2000) belonging to the contributed R libraries nlme, splines and lmesplines (Venables and Ripley, 2002), respectively. In this model, soil depth (parameterized as a b-spline with three degrees of freedom), year and N were set as fixed factors, while each lysimeters CNR depth profile was a random factor. 3. Results 3.1. Experimental conditions Summarized for each growing season, the mean air temperatures were similar in 1999 (12.9 ◦ C) and 2002 (12.6 ◦ C) and in 2000 (13.8 ◦ C) and 2001 (14.0 ◦ C). The mean air temperatures were particularly lower from sowing (SO) to stem elongation (SE) in 1999 and 2002 (Table 2). Cumulative precipitation varied among the years to a greater extent than mean air temperature: from 428 mm in 2002 to 611 mm in 2001. The periods most affected by reduced precipitation, were from SO to SE in 2000 and from SO to the beginning of tillering (BT) in 2002. 3.2. Interannual variation in shoot growth and N off take Table 3 shows the dates of the principal growth stages. The longest growth period was observed in 1999 while the earliest time of all the developmental stages was observed in 2000. Anthesis (AN) occurred latest in 2002, resulting in the shortest grain filling period of the 3 years of the experiment.

Table 2 Mean air temperature (◦ C) and cumulative precipitation (mm) during the growth periods used in the interannual comparisons Mean air temperature (◦ C)

SO-BT BT-SE SE-AN AN-PM

Cumulative precipitation (mm)

1999

2000

2001

2002

1999

2000

2001

2002

6.9 13.3 15.4 17.7

8.0 15.6 15.3 15.1

8.7 15.1 15.5 15.9

7.2 8.0 15.0 15.6

121 193 144 177

91 22 163 255

203 78 148 180

33 143 132 119

SO: sowing, BT: beginning of tillering, SE: stem elongation, AN: anthesis and PM: physiological maturity.

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Table 3 Days after sowing of the reference growth stages used in interannual comparisons Year

Growth stage

1999 2000 2001 2002

Beginning of tillering, 20–22a

Stem elongation, 30–32a

Anthesis, 64–66a

Physiological maturity, 92+a

43 33 40 31

62 50 65 58

91 82 88 102

147 137 137 136

Values are averages of the two levels of N supply treatments. a BBCH-code.

Shoot N off take, measured at physiological maturity, was affected significantly (p < 0.05) by year and nitrogen supply (NS) and their interaction (Table 4). The interaction between year and NS also modified the effect of the single factors on the yields of the studied components of the shoot and the N concentration in the straw (Table 4). At LN, the N off take in 2000 (20 g m−2 ) was higher than in the other 3 years as a result of higher straw and grain yields and higher N concentrations. Straw and grain yields were highest in 2000 and lowest in 2001 (Table 5). The N concentrations of the straw (0.78%) and the grain (1.88%) were highest in 2000, too, but were intermediate in 2001 (Table 5). There were no differences in N off take by the shoot in 1999, 2001 and 2002 due to the opposite results for biomass yields and biomass N concentrations (Table 5). At HN, N off take (Table 5) was highest in 1999 (30 g m−2 ) and 2000 (32 g m−2 ) compared to 2001 (22 g m−2 ) and 2002 (17 g m−2 ). Although straw yields were rather low in 2000 and 2002, they differed to a lesser extent among years than grain yields (Table 5). N off take and grain yield had a similar rank order among years. Due to the significantly lower N concentration in the grains in 1999 (2.17%), N off take was slightly higher in 2000. As a result of the differences in grain yield the N off take in 2000 and 2002 differed by approximately 32% (Table 5).

with year showed the same systematic effect on MNR but not on the other two parameters. NS changed 50/75MNR, mainly in the topsoil (0.05–0.10 m), but not 50MNR. Y × NS influenced 50MNR (0.05–0.15 m) and 50/75MNR (0.10–0.30 m) in the topsoil, but not in deeper soil layers. Due to these contrasting results, two soil depths from the topsoil (0.10 and 0.20 m) and two from the subsoil (0.45 and 0.80 m) were selected for the description of root growth. With the exception of 2000, HN tended to increase CNR (higher MNR) and to prolong root growth (higher 50MNR and 50/75MNR) at all soil depths compared to LN (Table 7). MNR was higher between 2 and 244% at HN depending on the soil Table 5 Means of selected parameters of the shoot as affected by year at two levels of N supply N supply (kg N ha−1 )

Year

20 Shoot N off take 1999 2000 2001 2002

270

(g m−2 ) 9.74 b 20.16 a 7.23 b 8.22 b

29.75 a 32.03 a 22.34 b 16.97 c

Grain yield (g m−2 ) 1999 2000 2001 2002

471 b 795 a 216 c 279 bc

938 a 885 a 587 b 459 c

Table 6 shows the effects of year and NS and their interaction at different soil depths on the parameters of the logistic equation used to fit CNR. Year affected the three parameters of the logistic equation at almost all soil depths. NS and its interaction

Straw yield (g m−2 ) 1999 2000 2001 2002

786 b 871 a 455 c 555 c

1301 a 1051 b 1216 b 996 c

Table 4 Effects of year (Y) and N supply (NS) and their interaction on shoot N off take and grain and straw yields and N concentrations of spring wheat at harvest in the years of the experiment

Grain N concentration (%) 1999 2000 2001 2002

1.58 b 1.88 a 2.29 a 2.22 a

2.17 b 2.39 a 2.73 a 2.67 a

Straw N concentration (%) 1999 2000 2001 2002

0.29 b 0.78 a 0.47 ab 0.35 b

0.71 a 1.02 a 0.52 ab 0.47 b

3.3. Interannual variation in the time course of root growth

Factor

Grain yield

Straw yield

Grain N

Straw N

Shoot N off take

Y NS Y × NS

***

***

***

***

***

***

***

***

***

***

***

***

ns

*

***

ns: non-significant at the 0.10 probability level. *, ***Significant at the 0.05 and 0.001 probability level, respectively.

Means followed by the same letter are not significantly different (p < 0.05) according to pair-wise T-tests.

J.M. Herrera et al. / Europ. J. Agronomy 26 (2007) 317–326

321

Table 6 Effects of year (Y) and nitrogen supply (NS) and their interactions on the parameters of the logistic equation of root development of spring wheat at different soil depths Soil depth (m)

Parameters of the logistic equation MNR Y

p-Values of the F-test 0.05 0.10 0.15 0.20 0.25 0.30 0.45 0.60 0.80 1.00

50MNR Y × NS

NS

50/75MNR

Y

NS

Y × NS

Y

ns ns ns ns

** **

***

**

*

***

†

***

***

†

†

ns

*

***

*

**

**

**

ns

ns

ns

**

†

*

**

**

**

†

ns ns ns ns

***

**

**

***

**

*

*

***

*

**

**

***

ns

ns

ns

ns

NS

Y × NS

**

*

ns

*

*

***

**

***

***

ns ns ns

**

ns ns

**

**

***

**

*

†

***

ns ns

ns ns ns

***

†

*

ns ns

†

*

ns ns ns ns

MNR: maximum cumulative number of roots cm−2 , 50MNR: time at which the cumulative number of roots cm−2 is 0.5 of the maximum, 50/75MNR: time lapse between 50MNR and the time at which the cumulative number of roots cm−2 is 0.75 of the maximum, ns: non significant at the 0.10 probability level. †, *, **, ***Significant at the 0.10, 0.05, 0.01 and 0.001 probability level, respectively.

depth and year. The effect of HN on 50MNR was smaller (−8 to 35%), while its influence on 50/75MNR varied from −32 to 103% (Table 7). At LN, the most intense root growth (MNR) in all the soil layers (3.11 at 0.80 m to 6.32 roots cm−2 at 0.10 m; Table 7) occurred in 2000. The values were quite high in 2001, although rarely significantly higher than in 1999 or 2002. No systematic

pattern was observed for the other years. Significant differences in the time course of root growth, as estimated by 50MNR, were observed at all depths and ranged from 41.8 (at 0.45 m in 2001) to 68.8 d (0.80 m in 2002). 50MNR was lower in 2000 in most of the soil profile (Table 7). The time course of root growth in the soil layers varied strongly from year to year: it increased continuously from 0.10 to 0.80 m (1999) and from 0.20 to 0.80 m

Table 7 Means of the parameters of the logistic equation of root development of spring wheat at different soil depths and nitrogen supplies as affected by year Year

Parameters of the logistic equation MNR (roots cm−2 )

50MNR (days)

50/75MNR (days)

20a

270a

20a

270a

20a

270a

0.10 m 1999 2000 2001 2002

1.98 b 6.32 a 3.90 ab 2.54 b

4.78 4.91 6.47 4.62

43.82 b 57.33 a 57.61 a 56.96 a

59.16 50.28 57.62 55.62

6.95 11.26 11.32 9.98

13.22 ab 14.95 a 12.54 b 7.16 b

0.20 m 1999 2000 2001 2002

2.22 ab 3.32 a 2.72 a 1.38 b

2.46 b 2.71 b 6.23 a 3.09 b

45.87 b 47.27 b 57.40 a 56.31 a

53.61 b 43.89 b 61.13 a 60.22 a

5.05 b 14.63 a 12.69 b 7.93 b

10.27 b 9.24 bc 14.61 a 5.77 c

0.45 m 1999 2000 2001 2002

1.01 c 3.39 a 2.67 ab 1.96 b

1.58 b 2.75 a 3.47 a 3.23 a

60.42 a 41.84 b 61.07 a 62.68 a

68.42 a 43.57 b 56.41 ab 66.49 a

12.69 a 2.64 c 13.54 ab 3.68 bc

13.45 a 2.89 c 11.86 ab 4.20 bc

0.80 m 1999 2000 2001 2002

1.82 3.11 1.23 2.08

1.86 b 2.79 ab 4.23 a 3.04 ab

66.23 a 48.45 b 58.58 ab 68.83 a

68.65 a 49.81 b 63.12 ab 72.07 a

9.33 a 5.19 b 7.56 ab 2.98 b

10.40 a 3.58 b 8.95 ab 2.44 b

MNR: maximum cumulative number of roots cm−2 , 50MNR: time at which the number of roots cm−2 is 0.5 of the maximum, 50/75MNR: time lapse between 50MNR and the time at which the cumulative number of roots cm−2 is 0.75 of the maximum. Means followed by the same letter are not significantly different (p < 0.05) according to pair wise T-tests. a N supply (kg N ha−1 ).

322

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Table 8 Means of the percentage of total roots of spring wheat developed by anthesis (PRA) at different soil depths as affected by year and nitrogen supply Factor

Soil depth (m)

Significance levels of the F-tests Y NS Y×N Year

1999 2000 2001 2002

0.10

0.20

†

†

**

*

*

ns ns

*

ns

ns

*

†

0.45

0.80

Nitrogen supply (kg N ha−1 ) 20

270

20

270

20

270

20

270

95 91 93 98

86 91 90 98

96 90 93 99

95 88 90 99

78 c 97 a 83 bc 95 ab

85 c 97 a 91 bc 99 ab

87 b 97 a 84 b 88 b

81 97 83 96

ns: non significant at the 0.10 probability level. Means followed by the same letter are not significantly different (p < 0.05) according to pair wise T-tests. †, *, **Significant at the 0.10, 0.05 and 0.01 probability level, respectively.

(2002), it decreased continuously from 0.10 to 0.45 m (2000) or it remained approximately constant at all soil depths (2001). The second parameter reflecting the time course of root growth, 50/75MNR, showed a single significant difference among years in the topsoil: 50/75MNR was higher in 2000 at 0.20 m (14.6 d) than in 1999 (5.1 d), 2001 (12.7 d) and 2002 (7.9 d). In the subsoil 50/75MNR was lower in 2000 (2.6 d at 0.45 m and 5.2 d at 0.80 m) and in 2002 (3.7 and 3.0 d) compared to 1999 (12.7 and 9.3 d) and 2001 (13.5 and 7.6 d). Overall, in the period during which 75% of the total CNR (50MNR + 50/75MNR) grew, root growth in the topsoil slowed down earlier in 1999 and in the subsoil in 2000, while the time course of root growth was similar for 2001 and 2002. At HN, the MNR was highest in 2001 (in contrast to the LN treatment, which had highest values in 2000). However, most of the differences among the years 2000, 2001 and 2002 at HN were not significant. The MNR was lowest in 1999 at all depths except for 0.10 m. The shortest period of root growth, indicated by a significant effect on 50MNR, was in 2000 at all the studied depths, while the longest period of root growth differed among the years in each soil layer. 50/75MNR showed the most systematic differences among the years. The smallest 50/75MNR was observed in 2002 at each depth except 0.45 m; below 0.10 m the values were similar than in 2000 (Table 7). The period of root growth was shortest at 0.10 m in 2002 and at the other soil layers in 2000 (Table 7). Although 75% of total CNR had always grown before anthesis (AN), root growth in spring wheat continued after anthesis, as indicated by the percentage of total CNR grown by anthesis (PRA, Table 8). The PRA at different soil depths ranged from 78% (0.45 m, LN, 1999) to 99% (0.45 m, HN, 2002). PRA was marginally affected by year in the topsoil and significantly affected by year in the subsoil. At 0.45 m, the mean PRA for both NS levels was higher in 2000 (97%) than in 1999 (81%) and 2001 (87%). At 0.80 m, the effect of year on PRA was modified by NS: significant differences among the years were observed only at LN, where the PRA was highest in 2000 (97%) compared to 1999 (87%), 2001 (84%) and 2002 (88%). Although, NS sig-

nificantly affected the PRA in most of the soil profile, the effect of NS differed in the topsoil and the subsoil: at 0.10 m the PRA was higher at LN, while at 0.45 and 0.80 m the PRA was higher at HN (Table 8). 3.4. Interannual variation in the vertical patterns of root growth Table 9 shows the results of the statistical analysis of the vertical patterns of CNR, as influenced by year and NS; the resulting fits are plotted in Figs. 1 (BT) and 2(SE, AN and PM). The model explains approximately 80% (BT), 73% (SE), 65% (AN) and 62% (PM) of the variability in the data. At BT a similar pattern, resembling an exponential decrease with increasing soil depth, was observed in the 4 experimental years (Fig. 1). However, the vertical pattern was significantly modified by year. Differences were particularly evident between 2001 and 2002 (Fig. 1). BT was the only stage, at which the vertical pattern of CNR was not affected by NS. From SE to

Fig. 1. Fitted cumulative number of roots cm−2 of spring wheat in relation to soil depth at the beginning of tillering (BT) as affected by experimental years.

J.M. Herrera et al. / Europ. J. Agronomy 26 (2007) 317–326

323

Table 9 Effects of the piecewise parameter that describes the pattern of the cumulative number of roots cm−2 at the studied soil depths (bs(D)), year (Y) and the effects of nitrogen supply (NS) and their interaction on the cumulative number of roots cm−2 of spring wheat at four growth stages Growth stage

Full model p-Values of the F-test Intercept bs(D) Y NS bs(D):Y Y:NS bs(D):NS Y: bs(D):NS Model after backward selection p-Values of the F-test Intercept bs(D) Y NS bs(D):Y Y:NS bs(D):NS Y: bs(D):NS LRT Fixed factor effects R2 Adj R2 Random factor effects Random factor Variance function Variable R2 Adj R2

Beginning of tillering, loge a

Stem elongation, Noa

Anthesis, Noa

Physiological maturity, Noa

ns

***

ns

***

***

***

***

***

†

**

**

**

ns

*

**

*

***

***

**

*

ns ns ns

ns

**

**

**

†

ns

†

ns ns

– – – –

***

–

ns

***

***

***

***

**

†

**

*

*

***

***

***

**

– – – ns

***

***

**

***

*

–

– ns

–

**

*

***

0.71 0.70

0.71 0.68

0.60 0.56

0.57 0.52

Lysimeter varExp

varPower Fitted values

0.81 0.80

0.76 0.73

0.69 0.65

0.67 0.62

ns: non-significant at the 0.10 probability level. a Transformation. †, *, **, ***Significant at the 0.10, 0.05, 0.01 and 0.001 probability level, respectively.

PM the vertical pattern of CNR showed a complex conditioning by the studied factors, with two (SE and AN) and three (PM) significant factor interactions (Table 9). CNR usually decreases with soil depth, but changes in the CNR among growth stages vary throughout the soil profile and among the various treatments combinations. At HN, but not at LN, and at most combinations of year by developmental stage, the maximum CNR in the soil profile was observed at 0.05 m and a linear decrease occurred down to at least 0.30 m. Unusual patterns of CNR were observed, too. For example, at LN at stem elongation in 2000, the CNR first increased down from 0.45 to 0.60 m before it decreased again, while at physiological maturity in 2002 there was almost no change in CNR throughout the soil profile. Therefore, the vertical pattern of CNR was irregular after BT (Fig. 2). 4. Discussion There was a significant annual effect in the total N off take by the shoot of spring wheat during the 4 years of the experiment (Table 4). These differences were associated with differences in

the straw and the grain yields (Table 5) and, to a minor extent, with differences in the N concentration of both shoot components (Table 5). The annual effect on most of the shoot and root parameters was modified by nitrogen supply (NS), indicating that such an effect differed depending on the level of N supply. With regard to the results in 2000, a probable increased N availability due to high mineralization, as a result of filling the lysimeters just before the start of the experiment, may have played a role. Thus, soil conditions in 2000 reflect an intensive disturbance but provide interesting information about the root growth of spring wheat. Shoot N off take at LN was similar in 1999, 2001 and 2002 (Table 5), even though the growth of the root system varied considerably in those years at LN (Table 7). In contrast, at HN, the shoot N off take, the grain yield (Table 5), as well as the number of roots (MNR, Table 7), varied among these 3 years. In 2001, N off take and grain yield were moderate but the highest number of roots were produced throughout the soil profile (MNR, Table 7). In 1999 (highest shoot N off take) and 2002 (least shoot N off take), the number of roots were similar in the topsoil, but

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Fig. 2. Fitted cumulative number of roots cm−2 of spring wheat in relation to soil depth at three growth stages and two N supplies as affected by experimental years. SE: stem elongation, AN: anthesis, PM: physiological maturity, LN: 20 kg N ha−1 , and HN: 270 kg N ha−1 .

significantly more roots were observed in the subsoil in 2002. Thus, when N supply was ample, the highest shoot performance was observed for the lowest number of roots cm−2 in the subsoil. However, in the topsoil at both N supplies and at all soil depths, a clear relationship was not found in LN between shoot performance and root growth. These results are in agreement with Barraclough (1986a), who did not observe a clear relationship between grain yield and root growth. This is surely due to the numerous and complex physiological processes, which determine shoot growth, root growth and nutrient uptake (see Gregory, 1994 for a review). These processes hinder the identification of relationships between the size of the root system and the shoot. There was substantial variation in the time course of root growth, as revealed by the analysis of the parameters of the

logistic equation (50MNR and 50/75MNR, Table 7). In 2000, in contrast to the other 3 years, roots in the subsoil grew during a shorter time (lower sum of 50MNR and 50/75MNR, Table 7) and this was associated with a lower root growth after anthesis than in 1999 and 2001 (Table 8). Root growth in 2000 was also shorter compared to 2002, but this effect was associated only with the period of exponential increase in the number of roots (50MNR, Table 7). The main differences in the time course of root growth among 1999, 2001 and 2002 were found in the period, during which the number of roots increased linearly (50/75MNR, Table 7). This indicates that the differences among these 3 years occurred at later developmental stages, suggesting that these differences had another cause than those in 2000. Barraclough and Leigh (1984) observed that N often increased the rate, at which root elongation occurs, but only

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between BT and AN. Campbell et al. (1977) explained that the capacity to sustain high rates of root growth for a longer period of time is the major and most consistent result of enhanced N availability. In our study, the maximum number of roots (MNR) increased in response to NS in most of the soil profile (Table 7), but the time course of root growth was influenced only in the topsoil (0.05–0.15 m for 50MNR and 0.10–0.30 m for 50/75MNR). The annual variability in the period, during which the number of roots increased linearly (50/75MNR) was slightly more pronounced at HN than at LN, since the differences at HN were observed in the subsoil and at 0.20 m and not only in the subsoil, as was the case at LN (Table 7). The higher variability at HN could be associated to the fact that in this treatment N was not supplied at once, but in a split application. The steady mineralization of N at LN (the only source of N after sowing) may have been less variable than the split input from the fertilizer at HN. Especially because this higher variability was observed in the parameter more associated to root growth at rather late growth stages. Additionally, the distribution of the fertilizer N is influenced by precipitation patterns, since water fluxes downward are necessary to distribute the fertilizer N in the profile, while mineralization can be assumed to be more uniform throughout the soil profile in the lysimeters. The differences in the subsoil at both NS levels suggest that the growth of roots in the subsoil at later growth stages may be influenced by annual differences among cropping seasons. The period during which the number of roots increased linearly (50/75MNR) was shortest in 2002 compared to 1999 and 2001 (Table 7); the values at HN and LN were similar (Table 7). As in 2000, root growth in 2002 also decreased earlier and the mean air temperatures were lower at the end of the growing season; in 2000 the precipitation was also lower at this stage (Table 2). The annual variation in the time course of root growth probably has important implications for the uptake and use of nutrients: early vigorous growth of roots was shown to increase N uptake (Liao et al., 2004), and quick elongation into deeper soil layers increase of the N uptake efficiency of various cover crops (Kristensen and Thorup-Kristensen, 2004). However, due to experimental constraints, more is known about the root growth of wheat at early stages than at late growth stages (Smit et al., 2000). Brouder and Cassman (1990) found for cotton that a cultivar, which is less sensitive to K deficiency, shows more intensive root elongation after peak bloom, indicating a less determinate pattern of root growth. MacKay and Barber (1986) gave a similar explanation of the greater yield of a maize cultivar that continued root growth after mid silk. Root growth after anthesis ranged from 1 to 22% of the total roots that grew throughout the growing season of spring wheat; the differences among years were significant in the subsoil (Table 8). NS also affected the PRA at 0.10, 0.45 and 0.80 m. The smaller PRA in the topsoil (Table 8) at HN suggests that a greater proportion of all the roots grew later in the growing season than at LN. From the PRA it is concluded that the root system of the spring wheat genotype Toronit is not determined, as opposed to the vegetative parts of the shoot (Causton and Venus, 1981), and new roots grow until physiological maturity. The importance of the roots grown after anthesis for grain yield and N uptake is still unclear. Wheat can take up N after anthesis (Oscarson et al.,

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1995; Barraclough, 1986b; Van Sanford and Mac Kown, 1987) and late applications of N usually increase the leaf N concentration and may delay leaf senescence (Baenziger et al., 1994). Slafer and Satorre (1999) hypothesized that the rapid leaf senescence that occurs during grain filling is due to a diminished root growth. Root growth after anthesis may be an important, not only in terms of resource uptake but also because of the late senescence of wheat roots. Roots that grow after anthesis may either compete with grains for carbon and N (Gooding et al., 2005) or it may be an important means of N retranslocation to the grain (Andersson et al., 2005). Gregory et al. (1978) observed that root growth, as measured by root length, occurred sequentially down the profile and that the roots deeper in the profile did not produce laterals before branching increased near the soil surface. Although, at coarse resolution, our study reveals a higher number of roots in the topsoil as compared to the subsoil, a clear and systematic distribution of roots throughout the soil profile was not observed (Table 7). Non-parametric modeling, used to describe the patterns of CNR with depth, described a relatively high proportion of the variance observed (Table 9). However, as the growing season progressed, the decreasing variation explained and the need for more parameters to describe these patterns was all indicative of an increase in variability from BT to PM. At BT the CNR decreased exponentially with increasing soil depth (Fig. 1) but at later developmental stages a clear pattern was not observed, especially at LN (Fig. 2). At BT an inverse relationship between the number of roots in the soil profile and the sowing date was evident. In 2001 and 2002, with the latest and earliest sowing dates, respectively, highest (2001) and lowest (2002) CNR was observed (Fig. 1). This may be associated with the higher temperatures in 2001 compared to 2002 (Table 2). The conclusion of Comfort et al. (1988), that N can decrease root exploration below 0.30 m was not supported by our data for any of the studied growth stages. NS affected the vertical pattern of root growth at SE (Table 9) but not at BT, indicating that there was relatively less sensitivity to N at early developmental stages (before SE). This is in agreement with the observations of other researchers (Wang and Below, 1992; Belford et al., 1987; Drew and Saker, 1975). A delayed effect of N on the root system will probably be found, especially in the present study, where the application of the N fertilizer was split into four doses between sowing an anthesis. This analysis of root growth provides basic knowledge of how variable the root response per year can be. Our results are in agreement with the plastic responses found under other experimental conditions (Hodge, 2004). These results will be useful for planning studies on the morphology and architecture of the root system and for interpreting experimental results by accounting for the potential variation among years. Forde and Lorenzo (2001) emphasized that the root system is highly sensitive to variations in the soil and that its response results optimizing its efficiency in the use of available resources. Taking site-specific variation in the field into account enable to increase N use efficiency significantly (Meisinger and Delgado, 2002). Thus, a

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