Field Crops Research 85 (2004) 213–236

Effect of N rate, timing and splitting and N type on

bread-making quality in hard red spring wheat
under rainfed Mediterranean conditions
Elvira Garrido-Lestachea, Rafael J. López-Bellidob, Luis López-Bellidoa,*
a
Departamento de Ciencias y Recursos Agrı́colas y Forestales, University of Córdoba, P.O. Box 3048, 14080 Córdoba, Spain
b
Departamento de Biologı́a y Producción de los Vegetales, University of Extremadura, 06071 Badajoz, Spain
Received 10 August 2002; received in revised form 27 June 2003; accepted 18 July 2003

Abstract

Three different experiments were designed to study the effects of N fertilizer rate, timing and splitting, and the response to
combined application of N and S fertilizer on the bread-making quality of hard red spring wheat (Triticum aestivum L.) over a 3-
year period in Vertisols under rainfed Mediterranean conditions. The following parameters were analyzed: grain yield, test
weight, grain protein content, gluten index and alveograph parameters (W: alveogram index; P: dough tenacity; L: dough
extensibility; P/L: tenacity–extensibility ratio). The N rate experiment included rates of 0, 100, 150 and 200 kg N ha
1 applied
on four different sites. The experiment was designed as a randomized complete block with four blocks. For the experiment on N
timing and splitting, a single rate of 150 kg N ha
1 was used, different fractions being applied at sowing, tillering and stem
elongation, at a single site; again, experimental design was a randomized complete block with four blocks. Finally, for the
experiment on the response to combined application of N and S fertilizer, a single fertilizer dose of 150 kg N ha
1 was applied in
two forms (urea þ ammonium nitrate and urea þ ammonium nitrosulfate) with one leaf application at ear emergence (zero,
25 kg S ha
1, 25 kg N ha
1, 25 kg S ha
1 þ 25 kg N ha
1 and 50 kg N ha
1), also at a single site, using a split-plot design with
four replications. Year-on-year variation in rainfall led to marked variations in wheat yield, grain protein content and bread-
making quality indices. A close correlation was observed between rainfall over the September–May period and both grain yield
and grain protein content (optimum values for both being recorded in the rainfall range 500–550 mm) as well as the alveogram
index. A negative correlation was observed between mean maximum temperatures in May and both test weight and alveogram
index (W). N fertilizer rate had a more consistent effect on bread-making quality than on grain yield. The highest values for grain
yield were recorded at an N rate of 100 kg ha
1, while maximum grain protein content values were recorded at 150 kg ha
1.
Application of half or one-third of total fertilizer N at stem elongation improved grain yield and grain protein content with
respect to applications at sowing alone or at both sowing and tillering. Increased N rates led to a considerable increase in W
values and to a reduction in the P/L ratio, thus improving dough balance, with a negative effect on the gluten index. Leaf
application of N at ear emergence only affected grain protein content and the W index. Soil or leaf application of S had no effect
on protein quality indices. The response of grain yield and grain protein content to fertilizer N differed from that reported for
temperate climates.
# 2003 Elsevier B.V. All rights reserved.

Keywords: Bread-making quality; N fertilizer; Hard red spring wheat

*
Corresponding author. Tel.: þ34-57-21-84-95; fax: þ34-57-21-84-40.
E-mail address: cr1lobel@uco.es (L. López-Bellido).

0378-4290/$ – see front matter # 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0378-4290(03)00167-9
214 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
1. Introduction
The environment has a profound impact on wheat
quality. This helps to explain why different parts of the
world produce such different types of wheat (Stone
and Savin, 2000). The Mediterranean climate, char-
acterized by increasing water deficit and thermal stress
during grain filling, may cause large fluctuations
not only in grain yield but also in grain protein content
and composition, with considerable effects on the
rheological properties of dough (Borghi et al., 1997;
López-Bellido et al., 2001). According to Johnson and
Mattern (1987), hard wheat is generally higher in
protein content than soft wheat, and is produced
mainly in low-rainfall regions, where yields are limited
by the production environment and tend to be lower
than wheat yields in regions with more favorable
production environments.
Wheat protein content is influenced by environ-
ment, cultivar, N fertilizer rate, timing and method
of N application, and the interactions between these
factors (Rao et al., 1993). These authors report that
strategies to control grain protein content appear to be
related not to the use of new cultivars but rather to the
management of other inputs, such as N.
The negative relationship between yield and grain
protein concentration is a reality in production sys-
tems. Nevertheless, this does not imply that higher
grain protein cannot be obtained at high-yield levels.
Both yield and protein can be increased simulta-
neously, up to a certain level, given an adequate N
supply to the plants and efficient translocation to
developing grain (Halloran, 1981; Bhatia and Rabson,
1987; Gate, 1995). The dilution effect is the most
consistent source of this negative relationship. Under
favorable growing conditions, starch and protein build
up simultaneously. Water stress and high temperature
during the grain-filling period hinder the conversion of
sucrose into starch but have less effect on protein
formation (Brooks et al., 1982). However, López-
Bellido et al. (2001) point out that the negative
correlation between wheat yield and quality indices
(protein content and dough quality), is less marked
than that reported by other authors, due to the con-
siderable year-on-year variation in weather patterns
typical of Mediterranean conditions.
Grain protein concentrations are the result of com-
plex interactions between N and water availability,
yield and temperature; this complexity often hinders
examination. In general, research has established soil
N availability and the amount and distribution of
precipitation as key environmental factors exerting
strong influence on protein content. Temperature, light
intensity, photoperiod, and the length of the grain-
filling period have also been shown to influence
protein content. According to Johnson and Mattern
(1987), those conditions leading to long grain-filling
periods (e.g. in northwestern Europe) should result in
well-filled grain with a low protein content. By con-
trast, under conditions of heat and drought such as
those in the US High Plains and the Mediterranean
regions, where the grain-filling period is shorter and
grain yields lower, wheat generally contains more
protein. Benzian and Lane (1986) found no significant
relationship between rainfall and nitrogen concentra-
tion. In contrast, in studies by López-Bellido et al.
(1998) grain protein content was found to be inversely
proportional to rainfall during the growing season
under rainfed Mediterranean conditions. According
to Gooding and Davies (1997), rainfall prior to grain
filling has been thought to encourage dilution of early
nitrogen reserves by vegetative proliferation in arid
environments. Later rainfall, during grain filling, can
cause a relative nitrogen dilution by extending leaf life
and maintaining photosynthesis and carbohydrate
translocation. Rao et al. (1993) maintain that the effect
of water stress on protein content is due mainly to its
influence on grain yield. Thus, grain protein content
might be expected to increase in drought conditions
and decline when rainfall is heavier. The same climatic
variables do not consistently exert the same effect on
grain protein content from one year to another, since
growth and grain filling in any given year and at any
given site are influenced by climate-related factors,
which in turn influence soil N availability and soil
water stress.
According to Stone and Savin (2000), temperature
induces a series of changes in the physiology of the
components of grain quality, which interact in com-
plex ways to change the quality apparent in the mature
grain. In many instances, grain nitrogen concentration
appears to increase with increasing temperatures and
reduced rainfall (Rao et al., 1993; Debaeke et al.,
1996; Gooding and Davies, 1997; Daniel and Triboi,
2000; Stone and Savin, 2000). Gooding and Davies
(1997) have reviewed the mechanisms postulated to
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
account for the effects of temperature: increasing
temperature can reduce carbohydrate accumulation
more than nitrogen accumulation as the rate of senes-
cence is increased, thereby reducing photosynthesis
and grain growth. The increase in the grain protein
percentage generally results in an increase in grain
quality, as measured by dough strength and related
measures, although some work has shown that as
temperature rises above a certain threshold (usually
ca. 30 8C) the positive relationship between grain
protein percentage and dough strength breaks down,
and may even become negative. This change in the
‘‘normal’’ relationship between grain protein per-
centage and dough strength is caused by a tempera-
ture-induced change in grain protein composition.
In many varieties of wheat, the gliadin:glutenin ratio
increases at temperatures above ca. 30 8C, although
this threshold is lower in heat-sensitive cultivars.
It appears that the gliadin:glutenin ratio increases
because accumulation of gliadin is reduced less by
elevated temperature than that of glutenin (Stone and
Savin, 2000).
N fertilizer rates and timing of application are a
decisive factor in the obtaining of high yields,
increased protein content and improved alveograph
parameters (Borghi et al., 1997; López-Bellido et al.,
1998). In semiarid regions, the efficiency of N ferti-
lizers depends greatly on the timing and intensity of
rainfall in relation to the cropping period and also on
the timing of fertilizer application. In the Mediterra-
nean agroclimatic zone, periodic soil water shortages
have a considerable impact on fertilizer efficiency.
Generally, the agronomic efficiency of N fertilizer in
Mediterranean climates is lower than that observed
in temperate areas (Craswell and Godwin, 1984).
According to López-Bellido et al. (1998), wheat yield
was influenced by the fertilizer N rate in the years
where the amount of rainfall exceeded 450 mm during
the growing season, under rainfed conditions. Data on
grain yield response to N fertilizers often vary mark-
edly from site to site and season to season, even within
a limited geographical region.
The greatest influence of nitrogen fertilizer on grain
quality is achieved through its effect on grain protein
concentration (Borghi et al., 1995; Debaeke et al.,
1996; Gooding and Davies, 1997). Nitrogen fertiliza-
tion contributes significantly to the increase in protein
content, especially when fertilizer rates satisfy the
215
requirements of both yield and protein formation
(Johnson and Mattern, 1987). Delayed application
of N favors protein build-up in the grain over an
increased yield (Sowers et al., 1994) and influences
the bread-making quality of the resulting flour (Ayoub
et al., 1994). These authors claim that split application
of fertilizer N increases grain protein concentration
and improves wheat baking quality. Nitrogen applica-
tions split between fall and spring, compared with a
single fall application, have produced higher yields
of hard red winter wheat. Several hard red winter
wheat studies have shown that N applications after the
boot stage can increase yield and, when moisture is
adequate for N uptake after application, grain protein
increases (Sowers et al., 1994). Delayed spring N
application can promote grain protein accumulation
more than yield (Fischer, 1993; Sowers et al., 1994;
Karen et al., 1994; Ottman et al., 2000). According to
Borghi et al. (1997), the beneficial effect of N split-
ting in the Mediterranean climate, may be related to
reduction of N losses and to a better translocation of
pre-anthesis assimilates to the grain. In contrast,
Gooding and Davies (1997), argue that in spring
wheat crops, or short season wheat in hot environ-
ments, the timing of N application for most efficient
utilization is often less critical as crop development
is much more rapid and long periods of slow growth
are less likely.
According to López-Bellido et al. (2001), N ferti-
lizer proved to be a key factor in determining bread-
making quality, and the best strategy available to the
farmer for optimizing wheat quality. However, the
influence of weather conditions and soil residual N
should be borne in mind when deciding on the addi-
tional fertilizer N to be used as a top-dressing with a
view to increasing yield and, particularly, enhancing
wheat protein content and bread-making quality.
Much of the research on the principles of efficient
fertilizer use has been carried out in the temperate
climates of developed countries. There is a need
for a major research effort to study the problems
of fertilizer use in semiarid climates such as the
Mediterranean region.
The aim of the present study was to determine the
effects of N fertilizer rate, N timing and splitting and
response to combined application of N and S fertilizer
on bread-making wheat quality in rainfed Mediterra-
nean conditions.
216 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236

2. Material and methods
The study was carried out in Andalucia, southern
Spain. Three different experiments were performed:
(1) N fertilizer rate, (2) timing and splitting of N
fertilizer and (3) types of fertilizer and leaf fertiliza-
tion. The three experiments were performed at sites
representative of the various Vertisols typical of the
Mediterranean region, where rainfed agriculture is
widespread (Table 1). The study was carried out over
3 years (1999–2000, 2000–2001 and 2001–2002). The
field experiments were conducted at the same sites for
3 years, in all cases using a 2-year wheat–sunflower
rotation.
The N rate experiment was conducted at four
different sites (Dehesilla, Santa Clara, Carrasco and
Saladilla) and was designed as a randomized complete
block, with four treatments: 0, 100, 150 and 200 kg
N ha
1 (applied 1/3 at sowing þ 1=3 at tillering þ 1=3
at stem elongation) and four replications (Table 1).
The area of each basic plot was 50 m2 (5 m  10 m).
The experiment on timing and splitting of N ferti-
lizer application to wheat was conducted at a single
site (La Reina) and was also designed as a randomized
complete block using the same plot size and number of
replications (Table 1). Ten N treatments were applied,
all at the same rate (150 kg N ha
1), and split in
various proportions between sowing, tillering and
stem elongation: 0–0–0, 150–0–0, 100–50–0, 100–
0–50, 75–75–0, 75–0–75, 50–100–0, 50–50–50, 0–
150–0, and 0–75–75 kg N ha
1.
The third experiment was also conducted at a single
site (Casalillas) and was designed as a randomized
complete block with a split-plot arrangement and four
blocks. Again, the area of each basic plot was 50 m2
(50 m  10 m). Main plots were application to soil of
a single dose of 150 kg N ha
1 (1/3 at sowing þ 1=3 at
tillering þ 1=3 at stem elongation), either in the form
or urea at sowing and ammonium nitrate as top-dres-
sing, or as urea at sowing and ammonium nitrosulfate
at top-dressing. Sub-plots were leaf application (at ear
emergence) of the following treatments: 25 kg S ha
1,
25 kg N ha
1, 25 kg S ha
1 þ 25 kg N ha
1, 50 kg N
ha
1 and untreated control.
The cultivar used was Gazul, a short-cycle semi-
early hard red spring wheat of medium-to-low height.
The cultivar displays good productivity and adapt-
ability, and very good flour-bread-making quality. The
preceding crop was sunflower at all sites. Every year,
wheat plots were also supplied with P fertilizer at a
rate of 65 kg P ha
1; the fertilizer was incorporated
into the soil. Soil available K was adequate.
Gazul wheat was sown in December, at all sites and
in all years, at a seeding rate of 150 kg ha
1, in 18 cm
wide rows. Wheat was harvested early in June each
year, using a 1.5 m wide Nursemaster elite plot com-
bine (30 m2 per plot). Grain protein content was
determined by near infrared reflectance spectroscopy,
using a Bran þ Luebbe InfraAlyzer 260 instrument.
Test weights (weights per unit of volume) were
obtained with a Schopper chondrometer. Wheat-grain
quality was evaluated using a Chopin alveograph in
accordance with the ICC 121 Standard Method of the
International Association of Cereal Chemistry (ICC,
1986). The alveograph was used to measure the
rheological properties of dough prepared from flour
and water under standard conditions. The dough was
formed into disc-shaped pieces that were inflated into
bubbles. The pressure variation inside each bubble
was recorded in graphical form as an ‘‘alveogram’’.
The area under the curve is proportional to the energy
required to cause the test piece (or dough bubble) to

Table 1
Soil properties at experiment sites (depth 0–30 cm)

Experiment Location Organic pH Sand Silt Clay EC CO3
P (mg K (mg NO3
-N

1
matter (%) (dS m ) (%) kg
1) kg
1) (mg kg
1)

N rate Dehesilla (Montemayor, Córdoba) 1.9 7.6 20 40 40 0.23 12.1 9.4 587 4.8
Santa Clara (Carmona, Sevilla) 1.1 7.7 32 32 36 0.18 12.0 16.2 430 6.2
Carrasco (Baena, Córdoba) 1.2 7.8 16 28 56 0.23 13.1 8.2 704 9.1
Saladilla (Santaella, Córdoba) 1.0 7.9 36 20 44 0.17 18.5 5.9 325 10.4
N timing La Reina (Córdoba) 1.0 7.9 28 28 44 0.24 25.8 6.9 305 8.5
N type Casalillas (Córdoba) 1.3 8.0 40 20 40 0.23 23.3 13.2 318 9.5
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
break (W is the alveogram index, in 10
4 J). The
maximum height of the curve provides an estimate of
dough tenacity (P denotes overpressure, in mm) and
its length is a measure of dough extensibility (L is
the abscissa at rupture, in mm) (Rasper, 1991). The
European ranking based on the alveogram index is as
follows: class E (improved wheat), W > 250  10
4 J;
class 1 (ordinary wheat for direct bread-making),
250  10
4 J > W > 160  10
4 J; class 2 (wheat
for biscuits), W < 160  10
4 J. To be appropriate
for bread-making, wheat should also have a tenacity-
to-extensibility ratio P=L < 1.
The gluten index was determined using the Gluto-
matic system according to ICC standards 137, 155 and
158 (ICC, 1986): dough is prepared from a flour
sample by adding a saline solution, and wet gluten
is subsequently washed to separate gluten from
washed-out components. When the gluten is centri-
fuged at 6000 rpm, the least adherent fraction passes
through a 600 m-mesh polyamide sieve.
Annual data for each parameter over the whole
2-year period were subjected to analysis of variance
(ANOVA), using a year-combined randomized com-
plete block design according to McIntosh (1983).
Treatment means were compared using Fisher’s
protected least significant difference (LSD) test at
P  0:05. LSDs for different main effect and interac-
tion comparisons were calculated using the appropri-
ate standard error terms following Gómez and Gómez
(1984). Correlations between some of the study para-
meters were also calculated. The Statistix v. 7.0
(Analytical Software, 2000) was used for this purpose.
3. Results and discussion
3.1. Weather conditions
Fig. 1 shows monthly rainfall and temperatures at
the six experimental sites over the 3-year study. Rain-
fall varied considerably more between sites in any
given year than over the 3 study years. Mean annual
rainfall for all sites recorded a maximum interannual
variation of 150 mm; 2000–2001 was the wettest year
(609 mm), followed by 2001–2002 (504 mm) and
finally 1999–2000 (459 mm). The same trend was
recorded at all sites as regards the wettest year, and
also—albeit only partially—with regard to the other
217
2 years (Fig. 1). Mean annual rainfall for the area over
the last 30 years is 584 mm; 1999–2000 and 2001–
2002 were therefore dry years and 2000–2001 was
normal. Inter-site variations in rainfall within a single
year displayed extreme values of 211 mm in 1999–
2000, 264 mm in 2000–2001 and 336 mm in 2001–
2002 (Fig. 1). This considerable spatial variation in
rainfall, consistently reflected in fluctuations in wheat
yield, is also a characteristic feature of the Mediter-
ranean climate, although less widely documented than
year-on-year variations in rainfall. The greatest dis-
tance between experimental sites was 120 km, in an
east–west direction.
Rainfall distribution also differed between years
and was very similar within each year for the different
sites (Fig. 1). In 1999–2000, a mean 40% (range 33–
43%) of total rainfall for all sites was recorded in
autumn, prior to wheat sowing; rainfall was scarce in
winter, 15% (range 12–20%); spring accounted for
44% (range 38–53%) of total annual precipitation. By
contrast, in 2000–2001 rainfall was low in autumn,
with a mean value of 18% (range 17–20%), and
abundant in winter with a mean value of 45% of total
annual rainfall (range 41–50%); mean spring rainfall
was 32% (range 28–37%). In 2001–2002, the rainfall
distribution pattern was more balanced, with a mean
27% in autumn (range 25–28%), 23% in winter (range
18–27%) and 38% in spring (range 33–43%).
Differences in temperature between sites over the
three study years were relatively modest (Fig. 1). The
highest aggregate temperatures were recorded in
2000–2001, both in winter and in spring (Fig. 1).
Mean winter temperatures ranged between 11 and
13 8C over the 3 years. Minimum winter temperature
ranged between 3 and 6 8C in 1999–2000; between
7 and 8 8C in 2000–2001 and between 5 and 6 8C
in 2001–2002. During the grain-filling period (April–
May) the mean temperature was 17 8C in 1999–2000
and 2001–2002, and 18 8C in 2000–2001 (Fig. 1). The
mean maximum temperature during this period varied
between 23 and 26 8C in 1999–2000; between 25 and
29 8C in 2000–2001 and between 24 and 26 8C in
2001–2002.
3.2. Grain yield
The response of wheat grain yield to N rate, timing
and splitting and type of N fertilizer varied depending
 218
E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
Fig. 1. Monthly and annual rainfall and mean maximum and minimum temperature for 3 years at different sites of Andalucia region (Spain).
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236 219

Table 2
ANOVA (mean square) of grain yield, test weight, grain protein, gluten index and alveograph parameters for a hard red spring wheat (cv.
Gazul) as affected by year, N rate, N timing and soil/leaf fertilization in 3 years (1999–2000 to 2001–2002)a

Source d.f. Grain yield  Test weight Grain Gluten Chopin alveograph parameters
104 (kg ha
1) (kg hl
1) protein (%) index (%)
W (10
3 J) P (mm) L (mm) P/L

N rate experiment
Year (Y) 2 2288*** 193.42*** 12.79*** 5034*** 193734*** 2442** 133357*** 3.38*
Location (L) 3 3466*** 54.34*** 33.59*** 545*** 15197 108 1184** 1.53
YL 6 1193*** 51.77*** 10.33*** 235*** 58775*** 4754*** 2138*** 3.90***
Error a 36 69 0.70 1.33 48 5496 440 261 0.58
N rate (N) 3 1733*** 107.71*** 134.00*** 424*** 259438*** 1070 14023*** 9.25***
YN 6 310*** 19.61*** 6.73*** 241*** 46575*** 976 1337* 0.88
LN 9 25 6.13*** 3.87*** 17 15419* 202 1213* 1.50*
YLN 18 41* 4.71*** 2.19*** 52** 8285 1001 876* 0.88
Error b 108 20 0.78 0.65 20 6265 538 475 0.70
N timing experiment
Year (Y) 2 1599** 79.72*** 58.20*** 7585*** 19460*** 488 9951*** 4.16**
N treatment (T) 9 589*** 1.91 2.83*** 54 5378** 776 897* 0.51
YT 18 171*** 0.91 0.80* 165*** 8411*** 862 1067** 0.60
Error 81 53 1.73 0.46 34 1785 512 418 0.36
Soil/leaf fertilization experiment
Year (Y) 2 2968*** 171.47*** 196.84*** 449** 68620*** 2225 306 4.34
Soil fertilization (S) 1 149 1.03 0.29 37 11252* 4600** 2332* 0.24
YS 2 201 1.46 0.28 36 2916 260 81 1.20
Error a 9 338 5.02 0.41 24 2059 363 402 1.15
Leaf fertilization (F) 4 30 0.62 4.74*** 50 31225*** 2529** 649 1.31
YF 8 43 5.29 0.40 22 4374 492 702 1.26
SF 4 18 7.87 0.41 56 1657 252 338 1.30
YSF 8 25 1.96 0.17 15 1685 384 152 1.23
Error b 72 23 3.23 0.29 25 3385 733 370 1.68
a
W: alveogram index; P: dough tenacity; L: dough extensibility.
*
Significant at the 0.05 probability level.
**
Significant at the 0.01 probability level.
***
Significant at the 0.001 probability level.

on the weather during the growing season. Interannual
variations were significant for all experiments (Table 2).
In the N rate experiment, grain yield differed sig-
nificantly between the three study years: the highest
value was recorded in 2000–2001 (4228 kg ha
1),
which was the wettest year at all four sites; followed
by 1999–2000 (3822 kg ha
1), when rainfall at 3 of the
4 study sites was greater than in 2001–2002; the latter
recorded the lowest yield (3051 kg ha
1) (Fig. 2a).
Year-on-year differences in rainfall in March and April
(a crucial period for the final phase of grain yield), also
had a significant effect on variations in yield (Fig. 1).
Grain yield also varied significantly between sites
(Fig. 2a): Dehesilla and Santa Clara recorded higher
values (4493 and 4331 kg ha
1, respectively), with no
significant difference between them, than Carrasco and
Saladilla (3240 and 2737 kg ha
1), which also showed
no significant differences. These differences in yield
may be attributed partly to variations in rainfall over
the three study years (Santa Clara recorded the highest
mean rainfall (540 mm) and Carrasco the lowest,
382 mm) and partly to differences in soil residual N
content prior to sowing, which varied for the experi-
ment as a whole between 31.3 and 88.6 kg ha
1, by
year, and between 36.5 and 87.8 kg ha
1, by site (soil
depth 0–90 cm) (unpublished data).
The fertilizer N rate had a significant effect on wheat
grain yield for the 3 years as a whole (Table 2 and
Fig. 2a) and at each trial site. Yield increased with
rising N rates only up to 100 kg N ha
1; no significant
220 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236

Grain yield Protein

7000 17
a
a a
a
6000 a 15
b b
b b
b
c
5000 13
Grain yield (kg ha-1)
A
A c

Protein (%)
A
A A
B A A
4000 11
B
C
3000 B B 9

2000 7

1000 5
2000

2001

2002

100

150

200
Carrasco
Dehesilla

Saladilla
Sta Clara

0
Year Location N rate (kg ha -1)

7500 16
a
a
b
abc
ab ab bc ab ab
7000 b bc 14
A c
c d AB A
A
6500 12
Grain yield (kg ha-1)

BC B
AB BC BC BC

Protein (%)
6000 10
C
B
5500 8

5000 6

D
4500 4

4000 2
2000

2001

2002

150-0-0

100-50-0

100-0-50

75-75-0

75-0-75

50-100-0

50-50-50

0-150-0

0-75-75
0-0-0

Year N timing (kg ha-1 )

7500 a 17
c
a 15
6500 a b
a cd c
d
b
Grain yield (kg ha-1)

13
c
5500
Protein (%)

A A 11
A A A A
4500 A A
A
9

3500 B
7

2500 5
2000

2001

2002

25N

50N
Nitrosulfate
Nitrate

25S

25N+25S

Year Soil fertilization Leaf fertilization (kg ha -1)

Fig. 2. Wheat grain yield and grain protein content as affected by year, location and N rate, N timing and type of fertilizer in three experiments
at Andalucia region, Spain (N timing: sowing–tillering–stem elongation; leaf fertilization: at ear emergence). Between years, locations and
within treatment means followed by the same letter are not significantly different at P < 0:05 according to LSD.
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
differences were recorded between 100, 150 and
200 kg N ha
1 (Fig. 2a). Similar results were obtained
by López-Bellido et al. (2000) in a long-term experi-
ment conducted under the same rainfed Mediterranean
conditions. The interactions year  location and year
N rate were highly significant and the triple interaction
year  location  N rate was significant (Table 2).
As reported by Craswell and Godwin (1984), the
response of yield to fertilizer N rate differed between
years. In the wettest year (2000–2001), yield responded
up to a rate of 150 kg N ha
1, whilst in drier years
a response was only recorded up to 100 kg N ha
1
(Fig. 3a). Moreover, the influence of year on yield
differed between non-fertilized and N-fertilized plots
(Fig. 3a); López-Bellido et al. (2001) attribute this to
year-on-year differences in the amount of soil available
nitrogen, due to the effect of the rainfall distribution
pattern on the mineralization of organic matter and
on crop residues. The inter-site differences in yield
analyzed above, prompted by variations in rainfall
and in the specific fertility of each site, would account
for the significance of the triple interaction year 
location  N rate.
Rainfall from September to May, which includes
the autumn fallow period prior to wheat sowing and
subsequent growth, showed the greatest correlation
with yield response to changes in the fertilizer N rate
(Fig. 4a), in comparison to other rainfall periods, such
as preceding fallow alone, pre-anthesis, grain filling or
the growth period as a whole. In the case of zero N,
maximum yield was obtained at around 450 mm rain-
fall in the September–May period; for N rates of 100,
150 and 200 kg ha
1, maximum yield was recorded
between 500 and 550 mm of rainfall. These results
agree with those reported by López-Bellido et al.
(1998, 2001), who also found that wheat yield was
only influenced by N rate when rainfall exceeded
450 mm during the growing season. The closeness
of the yield response curves for the three N rates tested
(100, 150 and 200 kg ha
1) indicates the absence of
any significant difference, as also reported by López-
Bellido et al. (2000) under rainfed conditions. The
decrease in wheat yield with application of rising N
rates where rainfall in the September–May period
exceeded around 550 mm has also been noted by
López-Bellido et al. (2001), who attribute it largely
to poor establishment and poor development of the
wheat crop during the vegetative phase, due to exces-
221
sive winter rainfall, which leads to waterlogging as a
result of the high clay content of Vertisols.
In the N fertilizer timing and splitting experiment,
performed at a single site (La Reina), grain yield
differed significantly between the three study years
(Fig. 2b). The highest yield was obtained in 1999–
2000 (6738 kg ha
1), when rainfall was lower than in
the other 2 years (Fig. 1); however, the difference in
yield with respect to 2001–2002 (6050 kg ha
1) was
not significant. The lowest yield was obtained in
2000–2001 (5474 kg ha
1), the wettest of the 3 years,
since rainfall was concentrated excessively in the
autumn–winter period, leading to poor establishment
of the crop and poor tillering due to waterlogging
(Fig. 2b). The timing and splitting of the
150 kg N ha
1 rate had a significant effect on wheat
grain yield over the 3 years as a whole (Table 2 and
Fig. 2b). The best grain yield response was obtained
when half or one-third of the total N fertilizer rate
(150 kg N ha
1) was applied at stem elongation; the
only exception to this was the treatment 50–50–
50 kg N ha
1 (Fig. 2b). Splitting of the total N rate
between sowing and tillering prompted a lower yield;
yield was lowest when the total rate of 150 kg N ha
1
was applied at sowing (except when zero N was
applied). Alcoz et al. (1993), Mossedaq and Smith
(1994) and Sowers et al. (1994) also report an increase
in wheat yield when fertilizer N is applied at the start
of stem elongation when moisture is adequate for N
uptake after application. The significance of the
year  N treatment interaction (Table 2) reflects the
effect of annual variations in the amount and distribu-
tion of rainfall on the uptake and efficiency of fertilizer
N at various stages in wheat growth (Fig. 5a).
Application of different types of fertilizer to soil and
of N and/or S fertilizer to leaf (third experiment,
conducted at Casalillas), had no significant effect
on wheat grain yield in any of the study years
(Table 2 and Fig. 2c). Gooding and Davies (1997)
have reported that amounts of N supplied as late
season foliar urea prompt an increase in grain protein
concentration rather than in grain yield. Application of
sulfur-containing fertilizers can sometimes increase
grain protein concentration. Significant differences in
yield were noted between years; the lowest yield was
obtained in 1999–2000, due to lower rainfall and poor
distribution over the growing season (little rain during
tillering and stem elongation).
222 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236

2000 2001 2002

6000 86
(a) (b)
5000 84

Test weight (Kg hl-1)

Grain yield (Kg ha-1)

82
4000
80
3000
78
2000
76
1000 74

0 72
0 100 150 200 0 100 150 200
N rate (kg ha-1) N rate (kg ha-1)

17 105
16
(c) 100
(d)
15 95
Gluten index (%)

14 90
Protein (%)

13 85
12 80
11 75
10 70
9 65
8 60
0 100 150 200 0 100 150 200
-1 -1
N rate (kg ha ) N rate (kg ha )

550
500
(e)
Alveogram index, W (x10-3J)

450
400
350
300
250
200
150
100
0 100 150 200
N rate (kg ha-1)

Fig. 3. Effect of year and N rate on wheat grain yield, test weight, grain protein content, gluten index, and alveogram index at Andalucia
region (Spain) over 3 years. Vertical bars represents LSD (P < 0:05).

3.3. Test weight weight values showed a direct correlation with grain
yield, i.e. weights were higher when yields increased:
Test weight was significantly influenced by year 80.8 kg hl
1 in 2000–2001 in the N rate experiment
in all three experiments (Table 2). Overall, annual test (Fig. 6a); 82.2 kg hl
1 in 1999–2000 in the fertilizer N
 5000 85
(a) (b)
4500 200N 83
100N
4000 81
Grain yield (kg ha-1)

Test weight (kg hl-1)

150N
3500 79 0N

E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236

3000 77

2500 75 100N
2000 2
0N: y = -0.12x + 106.2x - 20865; r²=0.34* 73 150N 0N: y = -0.0001x2 + 0.10x + 57.5; r²=0.1
100N: y = -0.07x2 + 66.5x - 12113; r²=0.38* 0N 100N: y = -0.0001x2 + 0.13x + 47.1; r²=0.55**
1500 150N: y = -0.07x2 + 73.1x - 13654; r²=0.40* 71 150N: y = -8E-05x2 + 0.10x + 49.4; r²=0.77***
200N: y = -0.05x2 + 57.0x - 10944; r²=0.48* 200N 200N: y = -0.0002x2 + 0.20x + 27.1; r²=0.75***
1000 69
300 350 400 450 500 550 600 300 350 400 450 500 550 600
September to May rainfall (mm) September to May rainfall (mm)

82 19
(c) (d)
80 0N 17 200N
Test weight (kg hl-1)

78 150N
15

Protein (%)
76 100N
13
74 100N
11
72 150N 0N: y = -0.0003x2 + 0.55x - 149.4; r²=0.06
100N: y = -0.0034x2 + 5.67x - 2261.5; r²=0.73*** 0N: y = -0.0001x2 + 0.11x - 13.39; r²=0.43*
150N: y = -0.0019x2 + 3.15x - 1253.5; r²=0.74*** 9 100N: y = -3E-05x2 + 0.02x + 12.74; r²=0.47*
70 0N
200N 150N: y = -0.0001x2 + 0.09x - 0.49; r²=0.77***
200N: y = -0.0014x2 + 2.50x - 1006.1; r²=0.81***
200N: y = -7E-05x2 + 0.04x + 9.96; r²=0.71***
68 7
770 790 810 830 850 870 300 350 400 450 500 550 600
Cumulative May mean maximum temperature September to May rainfall (mm)

Fig. 4. Relationship between grain yield, test weight and grain protein and September–May rainfall; and test weight and cumulative May maximum temperature according to N
rates fertilizer applied to wheat for 3 years at Andalucia region, Spain (0N: 0 kg ha
1; 100N: 100 kg ha
1; 150N: 150 kg ha
1; 200N: 200 kg ha
1).

223
224 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236

Fig. 5. Effect of year and N timing and splitting on wheat grain yield, grain protein content, gluten index and alveogram index at Andalucia
region (Spain) over 3 years (N timing: sowing–tillering–stem elongation). Vertical bars represents LSD (P < 0:05).

timing and splitting experiment (Fig. 7a); and 81.8 and
81.9 kg hl
1 in 2000–2001 and 2001–2002, respec-
tively, in the N fertilizer type experiment (Fig. 9a).
This direct relationship between test weight and grain
yield was observed at all four sites in the N rate
experiment (Fig. 6a); higher test weights were
recorded at Santa Clara and Dehesilla (80.3 and
79.6 kg hl
1, respectively), which also displayed higher
grain yields; conversely, lower test weights and lower
grain yields were obtained at Carrasco and Saladilla
(77.9 and 78.6 kg hl
1, respectively). López-Bellido
et al. (1998, 2001) report a consistent increase in
test weight with grain yield in rainfed Mediterranean
conditions.
N fertilizer rate influenced test weight, as reported
by Zentner et al. (1990), weights declining as the N
rate increased; differences in weights between the
different N rates were significant (Fig. 6a). The
year  location interaction was significant for test
weight, for the same reasons as for grain yield
(Table 2). The year  N rate, location  N rate and
year  location  N rate interactions were also sig-
nificant (Table 2). The decline in test weights at rising
N rates was affected both by variations in annual
rainfall (year  N rate interaction) (Fig. 3b) and by
specific environmental conditions at each site, espe-
cially in terms of rainfall and the level of soil residual
N (location  N rate interaction) (Fig. 8a). The triple
interaction year  location  N rate was significant
when the variables of the previous double interactions
were incorporated.
As for grain yield, total rainfall over the period
September–May, which covers the preceding fallow
period and the whole of the growing season, showed
the greatest degree of correlation with test weight at
different N rates, except for the zero N rate, when
regression was not significant (Fig. 4b). As rainfall
increased, test weights increased for all N rates, with a
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236 225

Fig. 6. Test weight, gluten index and bread-making quality of hard red spring wheat according to the Chopin alveograph parameters
(W: alveogram index; P: dough tenacity; L: dough extensibility; P/L: tenacity–extensibility ratio), as affected by year, location and N rate in a
experiment at Andalucia region (Spain). Between years, locations and within treatment means followed by the same letter are not significantly
different at P < 0:05 according to LSD.
226 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236

Fig. 7. Test weight, gluten index and bread-making quality of hard red spring wheat according to the Chopin alveograph parameters
(W: alveogram index; P: dough tenacity; L: dough extensibility; P/L: tenacity–extensibility ratio) as affected by year, and N timing and
splitting in a experiment at Andalucia region, Spain (N timing: sowing–tillering–stem elongation). Between years and within treatment means
followed by the same letter are no significantly different at P < 0:05 according to LSD.
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
considerable approximation of individual curves;
maximum values were obtained with rainfall between
500 and 550 mm. Unlike the findings of other studies
(López-Bellido et al., 2001), rainfall during the filling
period had less effect on the increase in test weight.
The sum of mean maximum temperatures during
the month of May also correlated significantly with
test weight for all N rate except zero N (Fig. 4c). The
regression analysis showed that as the cumulative
value of maximum May temperatures (end of grain
filling), test weights also increased, to maximum
values which differed as a function of the N rate
applied; as N rate increased, maximum test weight
values shifted towards the right (higher cumulative
temperature values) (Fig. 4c). This variation in beha-
vior as a function of N rate may be related to the
influence of temperature on protein formation and the
Fig. 8. Effect of location and N rate on wheat test weight, grain protein content, alveogram index and tenacity–extensibility ratio at Andalucia
region (Spain) over 3 years. Vertical bars represents LSD (P < 0:05).
227
accumulation of starch in the grain, as suggested by
Gate (1995) and Gooding and Davies (1997).
The timing and splitting of N fertilizer had no
significant effect on test weight (Fig. 7a). Unlike
the case of grain yield, fertilizer N applied at both
tillering and stem elongation failed to affect test
weight in comparison to the application of the same
N rate at sowing (150 kg ha
1). Similarly, the type of
N fertilizer applied to soil and leaf (third experiment)
had no influence on test weight (Fig. 9a).
3.4. Grain protein content
Grain protein content varied significantly between
years (Table 2), it was higher in 1999–2000 in all three
experiments: 14% in the N rate experiment; 14.7% in
the fertilizer timing and splitting experiment and
228 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
Fig. 8. (Continued ).
16.2% in the type of fertilizer experiment (Fig. 2).
The effect of the following 2 years (2000–2001 and
2001–2002) on grain protein content was unclear,
results were similar in the first experiment (Fig. 2a)
but contradictory in the second and third experiments
(Fig. 2b and c). Rainfall in April and May (the grain-
filling period) was considerably higher in 1999–2000,
when grain protein content was also greater for all
experiments as a whole (mean 174 mm for the six
sites; range 150–198 mm) than in 2000–2001 (40 mm;
range 28–78 mm) or 2001–2002 (81 mm; range 74–
88 mm). Similar results are reported by López-Bellido
et al. (2001), who found that heavy rainfall during
accumulation of protein in the grain (May) increased
protein content; other authors, however, report that
grain protein content increases under conditions of
drought or low rainfall (Gooding and Davies, 1997).
However, Gooding and Davies, like Rao et al. (1993)
argue that the mechanisms underlying the effect of
rainfall and soil moisture are complex, and that the
same climatic variables do not consistently have the
same effects at all site or in all years. Grain protein
concentrations are the result of interactions between N
and water availability, yield and temperature, whose
complexity in many cases hinders their examination.
The trial sites in the N rate experiment also recorded
significantly different grain protein contents: Dehe-
silla 13.8%, Santa Clara 13.5%, Carrasco 14.4% and
Saladilla 12.4% (Fig. 2a).
Grain protein content displayed a highly significant
response to N fertilizer rate, increasing with rising
N rates (11.2% zero, 13.5% 100 kg ha
1, 14.6%
150 kg ha
1 and 14.8% 200 kg ha
1) although there
was no significant difference between rates of 150
and 200 kg N ha
1 (Table 2 and Fig. 2a). Thus, maxi-
mum grain protein content was achieved with
150 kg N ha
1, when a rate of only 100 kg N ha
1
was required to achieve the maximum grain yield.
Similar findings are reported by Borghi et al. (1995),
who found that higher fertilizer N rates are generally
required to optimize protein content than to maximize
grain yield. However, and in contrast to the findings
of Gooding and Davies (1997), grain protein content
did not respond consistently to increases in N rate,
as it apparently does in other areas considered less
environmentally limiting for wheat. Consequently,
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236 229

Fig. 9. Test weight, gluten index and bread-making quality of hard red spring wheat according to the Chopin alveograph parameters
(W: alveogram index; P: dough tenacity; L: dough extensibility; P/L: tenacity–extensibility ratio) as affected by year, and type of soil
fertilization and leaf fertilization in a experiment at Andalucia region (Spain) (leaf fertilization: at ear emergence). Between years and within
treatment means followed by the same letter are not significantly different at P < 0:05 according to LSD.
230 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
from 150 kg N ha
1 upwards there is no increase in
protein levels and grain ceases to act as a sink for the N.
In this case, fertilizer N application is uneconomical,
since it prompts a luxury uptake of N, in excess of yield
requirements, and also gives rise to potential problems
of leaching and runoff of nitrates.
Year  N rate and location  N rate interactions
(Table 2) were significant, due largely to the difference
in behavior of grain protein content in N-free plots at
different sites and in different years, compared to plots
receiving fertilizer N at various rates (Figs. 3c and 8b).
Borghi et al. (1995) suggest that variations in the
amount of protein accumulated in grain on unfertilized
plots may results from inter-year (or inter-site) differ-
ences in soil available N, as indicated above under
grain yield.
The relationship between protein content and grain
yield for the 3-year study as a whole was far from
uniform. Behavior varied: in some cases, the highest
grain protein level was associated with the highest
grain yield (1999–2000, N fertilizer timing and split-
ting experiment) (Fig. 2b); in others, it was associated
with the lowest grain yield (1999–2000, type of N
fertilizer experiment) (Fig. 2c); elsewhere, intermedi-
ate situations were recorded, in which the highest
grain protein content was recorded for intermediate
grain yield, or intermediate grain protein content was
associated with medium to high grain yield; in some
15
14
Protein (%)
13
12
11
0 100
N rate (kg ha-1)
Fig. 10. Effect of N fertilizer on wheat grain yield and grain protein content. Vertical bars represent standard errors.
cases, low grain protein content was associated with
medium grain yield (Fig. 2). As a result, this study did
not consistently detect a negative relationship between
grain protein content and grain yield due to the effect
of N dilution, as reported by Halloran (1981), Bhatia
and Rabson (1987), Stoddard and Marchall (1990) and
Gate (1995). López-Bellido et al. (2001), in previous
experiments under Mediterranean conditions, found
that this negative relationship was less marked than
suggest by other authors, due to the considerable year-
on-year variation in weather patterns typical of Med-
iterranean areas. In contrast to the assertions of Toms
(1965), when the relationship between grain protein
content and grain yield is plotted as a function of N
rate (Fig. 10), there is an area (up to 100 kg N ha
1) in
which both grain yield and grain protein increase at a
rapid rate (slopes almost parallel); in a second area
(from 100 to 150 kg N ha
1) grain yield remains
practically stable, while grain protein content increases
slowly; in a third area (from 150 to 200 kg N ha
1)
both grain yield and grain protein content remain more
or less constant. This different pattern of response of
grain yield and grain protein content to fertilizer N may
be attributed to the shortage of water and to the high
temperatures typical of Mediterranean areas during
the grain filling stage. Both Gate (1995) and Gooding
and Davies (1997) suggest that moisture stress and
increased temperatures reduce grain carbohydrate
4500
Protein
4000
Grain yield
Grain yield (kg ha-1)
3500
3000
2500
2000
150 200
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
accumulation rather than N accumulation. This,
together with the lower agronomic efficiency of N
fertilizer typically found in Mediterranean though
not in temperate climates, would account for limited
wheat grain yield occurring in conjunction with high
grain protein content.
As for grain yield, rainfall from September to May
showed the greatest correlation with grain protein
content, as a function of N rate (Fig. 4d). Regression
analysis showed a difference in behavior between the
zero rate and the other N rates applied (Fig. 4d). At a
rate of zero N, grain protein content increased with
rainfall up to a maximum of around 450 mm, decreas-
ing slightly thereafter (Fig. 4d); at N fertilizer rates of
100, 150 and 200 kg ha
1, grain protein content was
generally greatest when rainfall was lowest, and
declined as rainfall increased due to the effect of N
dilution (Fig. 4d). In the absence of fertilizer N, soil
available N is not sufficient (under rainfed Mediterra-
nean conditions) to achieve high protein levels. How-
ever, as rainfall increases, protein content increases
more than grain yield, due to improved availability
and plant N extraction. By contrast, as reported by
Terman et al. (1969), the primary effect of fertilizer N
application is to increase grain yield in direct relation
to moisture availability, leading to N dilution in the
plant and a consequent reduction in grain protein
content. Previous studies by López-Bellido et al.
(2001) in the Mediterranean area have shown the
marked influence of rainfall during May (the period
when protein accumulation takes place in the grain) on
grain protein content, which was greatest with 80 mm
of rain and decreased thereafter. In the present study,
this relationship was significant only in the absence of
fertilizer N; regression analysis showed that grain
protein content was highest with only 50 mm of rain,
and decreased thereafter (y ¼
0:003x2 þ 0:31x þ
4:97; r 2 ¼ 0:78 ).
The timing and splitting of N fertilizer application
also influenced grain protein content, which was
always highest when half or one-third of the
150 kg N ha
1 rate was applied at stem elongation,
and in some cases when N was applied only at tillering
(Fig. 2b). Application of the whole 150 kg N ha
1 at
sowing prompted a significant decline in grain protein
content (Fig. 2b). A number of studies report that split
and late application of N promotes a build-up of grain
protein content rather than an increase in grain yield
231
(Sowers et al., 1994; Ayoub et al., 1994; Debaeke et al.,
1996). The year  treatment interaction was signifi-
cant, due to the influence of year on variations in grain
protein content (Fig. 5b).
The effect of fertilizer type (N and S) and method
of application (soil and leaf) was highly significant
(Table 2). Grain protein content increased when the
maximum leaf N rate was applied at ear emergence
(50 kg N ha
1) and also using the N and S combina-
tion (25 kg N ha
1 þ 25 kg S ha
1) (Fig. 2c); similar
findings are reported by Gooding and Davies (1997).
However, application of S either to the soil, as ammo-
nium nitrosulfate, or to the leaf at ear emergence did
not prompt any increase in grain protein content, in
contrast to the findings of these authors.
3.5. Gluten index
According to Vrkoc et al. (1995), gluten index
values depend 95% on crop location, 4% on year
and 1% on fertilization in Central European climates.
However, since the gluten index is a new and as yet
relatively unused parameter for wheat bread-making
quality, little information is available on the relationship
between the gluten index and other indices, or on the
influence of N fertilizer and environment on the gluten
index. In the present study, high gluten index values
were obtained in the 3 years (range 80.7–96.2%). Curic
et al. (2001) report optimum values between 75 and
90% for Central European cultivars. Overall, values
were higher in 2001–2002 in all three experiments; year
exerted a highly significant effect in all experiments
(Table 2 and Fig. 6b). Trial site also had a highly
significant effect in the N rate experiment (Table 2
and Fig. 6b). Perten et al. (1992) confirms that the gluten
index may vary considerably within the same cultivar,
while Curic et al. (2001) report that the considerable
variations recorded are due not only to cultivar differ-
ences but also to different climate conditions.
The gluten index decreased as N fertilizer rates rose
(from 89.9 to 83.4), although no significant differences
were recorded between 150 and 200 kg N ha
1 (Table 2
and Fig. 6b). Sip et al. (2000) report a similarly
negative effect of N fertilizer on this index. The
significant year  location, year  N rate and year
location  N rate interactions testify to the strong
influence of year and season on the gluten index
(Table 2 and Fig. 3d).
232 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
Mean temperature in May (the period of protein and
carbohydrate accumulation in the grain) was the cli-
mate parameter showing greatest correlation with glu-
ten index values at different fertilizer N rates (Fig. 11a).
The highest gluten index values, which were practically
the same for all N rates, were recorded at the lowest
mean temperatures (around 18.5 8C) (Fig. 11a). Gluten
index values declined with rising mean temperatures at
all N rates, although the decline was sharper at higher N
rates: linearly for the zero N rate, and quadratically for
the other N rates (Fig. 11a). According to Blumenthal
et al. (1991, 1993), high temperatures and low relative
humidity (typical of the Mediterranean climate) during
grain filling reduce gluten quality by accelerating grain
maturity, thus favoring production of gliadin at the
expense of glutenin; this reduces protein quality and
causes some weakening of the dough.
The timing and splitting of N fertilizer application
had no significant influence on the gluten index
(Table 2 and Fig. 7b). However, the year  treatment
interaction was significant due, as indicated earlier, to
the marked influence of environment. A greater dif-
ference was observed in 1999–2000 than in the other
2 years for gluten index values as a function of timing
and splitting of N fertilizer application (Fig. 5c). The
type of fertilizer (N or S) applied to soil and leaf
had no influence on the gluten index (Table 2 and
Fig. 9b).
A significant negative correlation was observed
between gluten index and grain protein content,
although the overall correlation coefficient for the 3
years as a whole was low (r ¼ 0:23 ; n ¼ 432). The
correlation was not significant in the N rate experi-
ment but was significant in both the timing and split-
ting experiment (r ¼
0:62 ; n ¼ 120) and the N
fertilizer type experiment (r ¼
0:24 ; n ¼ 120). As
indicated earlier, high temperatures and water stress
reduce gluten index values, but at the same may lead to
some grain protein enrichment by hindering the con-
version of sucrose into starch (Gooding and Davies,
1997). No significant correlation was observed
between the gluten index and the alveogram index
(W), in contrast to the findings of Curic et al. (2001).
3.6. Dough quality
All bread-making quality indices [alveogram index
(W), dough tenacity (P), dough extensibility (L) and
dough tenacity–extensibility ratio (P/L)] varied sig-
nificantly between years in the N rate experiment and
the N fertilizer timing and splitting experiment (except
P); W varied only in the fertilizer type experiment
(Table 2). The influence of year on bread-making
quality indices was less clear than for grain protein
content, with a marked disparity between values for
the three experiments (Figs. 6, 7 and 9). The alveo-
gram index (W) ranged between 216 and 351; dough
tenacity (P) between 88.1 and 111 mm; dough exten-
sibility (L) between 64.6 and 97.9 mm and dough
tenacity–extensibility ratio (P/L) between 0.99 and
1.91. The influence of trial site in the N rate experi-
ment was significant only for dough extensibility (L),
an index which appears to be more sensitive to varia-
tions in environmental conditions. The high alveo-
graph P/L values (mean 1.49 for the three experiments
over the three study years) compared to those reported
for Central Europe serve to confirm, according to
Borghi et al. (1997), the effect of high temperatures
on gluten strength.
The alveogram index (W) displayed a highly sig-
nificant positive correlation with grain protein content,
both over the study as a whole (r ¼ 0:56 ; n ¼ 432)
and for each of the individual experiments; similar
findings are reported in previous studies in the
Mediterranean area by López-Bellido et al. (1998,
2001). The alveogram index displayed a negative
correlation with test weight in all three experiments
(r ¼
0:53 ; n ¼ 432), and particularly in the N rate
experiment (r ¼
0:68 ; n ¼ 192).
Fertilizer N rate had a highly significant effect on all
bread-making quality indices except dough tenacity
(P) (Table 2). Increase in the N fertilizer rate prompted
increase in alveogram index (W) and dough extensi-
bility (L), and decreases in the dough tenacity–exten-
sibility ratio (P/L), thus improving dough balance
(Fig. 6). The value of W rose from 182 to 359, while
the P/L ratio fell from 2.25 to 1.33, as N rates rose
from 0 to 200 kg N ha
1, respectively, over the 3-year
period (Fig. 6). Borghi et al. (1995) have also reported
that increase in the N rate applied prompt an increase
in W, P and L values, improving the dough balance and
the shape of the alveogram. According to Gate (1995),
changes in these parameters may be due to an impaired
N/S ratio, which affects synthesis of sulfur-containing
aminoacids and the formation of some gliadins.
Thus, the effect of N fertilizer was more consistently
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236 233

105
0N: y = -2.42x + 139.45; r²= 0.52**
100N: y = 2.65x² - 108.0x + 1185.1; r²= 0.86***
100
150N: y = 2.84x² - 117.3x + 1292.3; r²= 0.61**
200N: y = 3.8x² - 156.8x + 1694.1; r²= 0.76***
95
Gluten index (%)

90 0N

85 100N

80 150N
200N
75
18 18.5 19 19.5 20 20.5 21
May mean temperature (ºC)

570
0N: y = -0.0075x² + 6.60x - 1236.8; r²= 45*
520 200N 100N: y = 0.0029x² - 3.16x + 1091.1; r²= 0.38*
150N: y = -0.0015x² + 0.46x + 400.4; r²= 0.50**
470
Alveogram index, W (x10-3J)

200N: y = 0.0014x² - 2.57x + 1233.6; r²= 0.63**

150N
420
370
100N
320
270
220
170
0N
120
70
300 350 400 450 500 550 600
September to May rainfall (mm)
800
0N: y = -1.95x² + 116.90x - 1523.7; r²= 0.33
100N: y = -7.75x² + 418.2x +5885.5; r²= 0.65**
200N 150N: y = 9.59x² - 504.0x + 6893.8; r²= 0.49*
650
Alveogram index, W (x10-3J)

200N: y = 5.637x² - 338.7x + 5354.6; r²= 0.66**

150N
500
100N

350

200
0N

50
21 22 23 24 25 26 27 28
May mean maximum temperature (ºC)

Fig. 11. Relationship between gluten index and alveogram index and May mean temperature, September–May rainfall and May mean
maximum temperature according to N rates fertilizer applied to wheat for 3 years at Andalucia region, Spain (0N: 0 kg ha
1; 100N:
100 kg ha
1; 150N: 150 kg ha
1; 200N: 200 kg ha
1).
234 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
favorable on wheat quality than on wheat grain yield
(López-Bellido et al., 2001).
The year  N rate location was significant for W and
L, while the location  N rate interaction was signifi-
cant for W, L and P/L (Table 2). The triple interaction
year  location  N rate was significant for L. In
contrast, P displayed no interactions. This again sug-
gests that dough extensibility (L) is more sensitive
to the environment than dough tenacity (P), which
appears to be more closely linked to the cultivar.
Indeed, the gliadin fraction is more responsible for
dough extensibility, whilst the glutenins are more
closely linked to tenacity; as indicated earlier, gliadin
production is increased by periods of high temperature
stress during grain filling. The significance of the
interaction is due to the varying behavior of W as a
function of N rate in 2002, together with disparate
values for W obtained with the zero N rate compared to
other rates (Fig. 3e). As is the case for protein, Borghi
et al. (1995) report that variations in quality indices on
unfertilized plots may be due to year-on-year variation
in the amount of soil available N. The same explana-
tion can be adduced to account for the significance of
the location  N rate interaction for the P/L ratio;
these variations were found for the zero N rate at all
sites (Fig. 8c).
Rainfall from September to May showed the great-
est correlation with the alveogram index (W), as a
function of N rate (Fig. 11b). As was the case for
protein content, regression analysis showed that the
zero N rate prompted a difference in behavior com-
pared to other N rates, at which W values were highest
when rainfall was lowest, declining as rainfall
increased; the decline was sharper, however, than that
observed for grain protein. As for grain yield, rainfall
from September to May showed the greatest cor-
relation with protein content (Fig. 11b). The close
correlation between grain protein content and the
alveogram index, also reported by Martin and Taureau
(1992), would account for the similar behavior of the
two parameters as a function of rainfall over the period
covering preceding fallow and the whole of the grow-
ing season.
Mean maximum temperatures during May also
displayed a significant correlation with the alveogram
index for all N rates except the zero rate; W values
declines with rising maximum temperatures (Fig. 11c).
As indicated earlier, high temperatures and low
relative humidity during grain filling prompt a weak-
ening of dough due to accelerated grain maturity,
which modifies the gliadin:glutenin ratio.
Timing and splitting of N fertilizer application
significantly influenced only the alveogram index
(W) and dough extensibility (L) (Table 2). Significant
differences were recorded for W between the zero N
rate (W ¼ 192) and the other N rates, with different
values for split application of the 150 kg N ha
1 rate
(range 228–261); there were no significant difference
between the various split applications (Fig. 7c). The
highest values for L were recorded when N was
applied at stem elongation and/or tillering, although
this was not always the case (Fig. 7e). Thus, splitting
and timing of N rate application did not display a clear
significant influence on alveogram indices, of the sort
recorded for grain protein content; similar findings are
reported for Mediterranean climates by Borghi et al.
(1997), although other authors disagree, including
Ayoub et al. (1994) and Le Souder et al. (1994).
Hucklesby et al. (1971) report that split application
of N influences the protein quantity but not quality.
The type of fertilizer (N or S) applied to soil had a
significant effect on all alveogram indices except the
dough tenacity–extensibility ratio (P/L) (Table 2).
Leaf fertilization also significantly affected the alveo-
gram index (W) and dough tenacity (P) (Table 2). At
equivalent N rates, values for W were higher when
ammonium nitrate was applied to soil than when
ammonium nitrosulfate was applied (Fig. 9c). Simi-
larly, W values increased with rising leaf N fertilizer
rates, whereas application of S fertilizer had no effect
(Fig. 9c). Dough tenacity (P) also displayed higher
values with application of ammonium nitrate to soil
than with leaf application of N (25 and 50 kg N ha
1);
values decreased with a single application of S fertilizer
(Fig. 9c). By contrast, dough extensibility (L) recorded
higher values with ammonium nitrosulfate, and was
not influenced by leaf application of N or S (Fig. 9e).
Consequently, the application of sulfur-containing fer-
tilizers had no evident effect on protein quality indices
as reported by Gooding and Davies (1997).
4. Conclusions
Year-on-year variations in climate and especially
in rainfall, typical of the Mediterranean area, result in
 E. Garrido-Lestache et al. / Field Crops Research 85 (2004) 213–236
considerable variation in wheat yield, grain protein
content and bread-making quality indices. Rainfall
over the period September–May (which includes the
fallow period preceding wheat and the whole of the
wheat growing season) displayed close correlation with
grain yield, test weight, grain protein content and alveo-
gram index (W) for all fertilizer N rates assayed. During
that period, grain yield and test weight recorded
maximum values with rainfall between 500 and
550 mm, declining thereafter. In contrast, grain pro-
tein content and alveogram index recorded maximum
values when rainfall for the period was lowest.
The considerable and prolonged water storage capa-
city of Vertisols ensures water availability for wheat
throughout the growing season. The rainfed cropping
model found in this area, which includes wheat, is
highly stable and efficient, since rainfall between
September and May, which optimizes grain yield, is
virtually the same as the 30-year mean annual rainfall
(584 mm). The distribution of rainfall over the wheat
growing season is of vital importance; excessive win-
ter rain leads to waterlogging and therefore adversely
affects wheat growth and development (poor plant
establishment and poor tillering).
The increase in mean maximum temperatures in
May, at the end of the grain filling stage, had a negative
effect on test weight and alveogram index (W), and on
the gluten index. High temperatures and water stress in
spring, characteristic of the Mediterranean climate,
shorten the grain filling and maturing period, reducing
grain yield and increasing protein content. They also
appear to prompt changes in protein composition,
which may negatively affect gluten strength and
dough quality in Mediterranean areas in comparison
to temperate climates.
Fertilizer N rates had a more consistent effect on
wheat bread-making quality than on grain yield. Grain
yield recorded optimum values at an overall N rate of
100 kg N ha
1, whilst maximum grain protein content
was achieved with 150 kg N ha
1. High N rates are
uneconomical, and may lead to problems of leaching
and runoff of nitrates.
Application of half or one-third of the fertilizer N at
stem elongation improved grain yield and grain protein
content, compared to application of the same dose at
sowing alone or at sowing and tillering.
Increased N rates (up to 150 kg ha
1) prompted a
considerable increase in alveogram index (W) values,
235
and reduced the tenacity–extensibility ratio (P/L),
improving the dough balance. In contrast, rising N
rates had a negative effect on the gluten index.
Leaf application of N in the form of urea at ear
emergence only affected grain protein content and the
alveogram index (W), but not grain yield. By contrast,
neither application of sulfur-containing fertilizer to
soil nor leaf application of sulfur at ear emergence had
any effect on protein quality indices.
The classical negative correlation between wheat
yield and grain protein content was not found, due to
the considerable year-on-year variation in weather,
typical of the Mediterranean area. The joint response
of grain yield and grain protein content to N fertilizer
in this climate differed from that reported for tempe-
rate climates. High temperatures and water shortage
during the filling stage increase the rate of senescence,
thus limiting photosynthesis and grain growth, and
reducing grain accumulation of carbohydrates rather
than of N.
Acknowledgements
This work was funded by FEDER Programme
and the Spain’s Plan Nacional I þ D (Proyects
1FD1997-0228 and AGL2001-2549). The authors
thank Assistant Technicians Joaquı́n Muñoz and
Auxiliadora López for their invaluable help and coop-
eration in the laboratory and field work.
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