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European Journal of Agronomy 155 (2024) 127119
Available online 29 February 2024
1161-0301/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Competition for light and nitrogen with an earlier-sown species negatively
affects leaf traits and leaf photosynthetic capacity of maize in
relay intercropping
Bei Dong
*
, Zishen Wang , Jochem B. Evers , Tjeerd Jan Stomph, Peter E.L. van der Putten,
Xinyou Yin , Jin L. Wang , Timo Sprangers , Xuebing Hang , Wopke van der Werf
Centre for Crop Systems Analysis, Wageningen University & Research, 6708 PE, Wageningen, the Netherlands
ARTICLE INFO
Keywords:
Interspecic competition
Light distribution
Leaf photosynthetic capacity
Leaf traits
Maize-faba bean intercrop
Maize-wheat intercrop
ABSTRACT
Mixing crop species in intercrops often results in yield advantages but the underlying processes are not
completely understood. Increased resource capture in intercrops, particularly of light and nutrients, has been
frequently demonstrated, but there is less information on the effect of intercropping on the photosynthetic ca-
pacity of leaves and on the leaf traits related to photosynthesis. Here we determine whether photosynthetic
capacity and associated leaf traits are enhanced in intercropped maize (Zea mays L.), a species frequently used in
intercrops. We determined leaf photosynthetic capacity (A
1800
) and leaf traits of maize leaves in different canopy
layers and at different growth stages in relay strip intercrops with spring wheat (Triticum aestivum L.) or faba
bean (Vicia faba L.) and in the maize sole crop. We also measured the distribution of photosynthetically active
radiation (PAR) in the canopies. Intercropping with wheat or faba bean resulted in larger specic leaf area (SLA;
thinner leaves), lower specic leaf nitrogen (SLN), and lower A
1800
of maize leaves during vegetative growth, and
differences were larger for maize intercropped with faba bean than wheat, consistent with stronger shading by
faba bean than wheat. After the harvest of companion species, maize leaves received more light in the two in-
tercrops than in the sole maize crop, but this did not result in increases in leaf N concentration, SLN, and A
1800
.
Results indicate that shading and lower leaf N caused by relay intercropping maize with an earlier sown species
negatively affected leaf photosynthetic capacity of maize leaves. The yield increase of maize in relay intercrops
was not due to a higher leaf photosynthetic capacity. Options for mitigating or overcoming these negative
intercropping effects are discussed.
1. Introduction
Plant production is driven by the photosynthetic conversion of at-
mospheric CO
2
to structural plant mass, and this process is supported by
light energy. Productivity increases can result from greater light capture
and (or) from higher light conversion efciency (Monteith, 1977;
Keating and Carberry, 1993). Mixed stands are usually more productive
than would be expected on the basis of the productivity of pure stands of
the species and their mixing proportion, resulting in a positive net
biodiversity effect (Loreau and Hector, 2001). This is true both in nat-
ural systems (Isbell et al., 2017; Tilman et al., 2001) and agricultural
production systems (Li et al., 2020b,c, 2023; Xu et al., 2020). It is also
well established that intercropping can result in enhanced light capture
compared to pure stands (Gou et al., 2017; Zhang et al., 2008). However,
there is less information on how leaf photosynthetic capacity is altered
in intercropping.
Leaf photosynthetic capacity differs between individuals of the same
species in a stand and between different leaves on the same plant ac-
cording to their position and age (Anten and Hirose, 2003). Compared
with shaded leaves, leaves that are well exposed to the sun have a higher
nitrogen (N) content per unit leaf area and higher leaf photosynthetic
capacity (Lambers et al., 2008; Walters, 2005). Such leaves are thicker,
and they have a lower specic leaf area (SLA) and a greater number of
chloroplasts per unit leaf area than shaded leaves (Evans and Poorter,
2001; Oguchi et al., 2003; Pengelly et al., 2010; Poorter et al., 2009).
Maize (Zea mays L.) is frequently used in species mixtures, and maize
often contributes substantially to intercropping yield advantages, more
than other species do (Li et al., 2020c, 2023). It is therefore relevant to
* Corresponding author.
E-mail addresses: bei.dong@wur.nl, dongbei44@gmail.com (B. Dong).
Contents lists available at ScienceDirect
European Journal of Agronomy
journal homepage: www.elsevier.com/locate/eja
https://doi.org/10.1016/j.eja.2024.127119
Received 31 May 2023; Received in revised form 6 February 2024; Accepted 6 February 2024
European Journal of Agronomy 155 (2024) 127119
2
understand the physiological response of maize to intercropping. To
date, studies on the response of maize leaf photosynthesis to inter-
cropping have focused on changes in actual rate of leaf photosynthesis
(Liu et al., 2018), or the rate of leaf photosynthesis under articial light
at lower than saturated light levels (Nasar et al., 2020, 2021, 2022; Yin
et al., 2021). These studies did not elucidate photosynthetic capacity of
maize leaves in the eld because the photosynthetic capacity is
expressed only after a leaf is well adapted to full light, allowing the leaf
sufcient time to open the stomata to completely match the CO
2
demand
of a well-lit leaf. Previous studies also lack information on the articial
light level and the adaptation time used to obtain leaf photosynthetic
rate (Feng et al., 2020; Li et al., 2020d; Ma et al., 2020; Yang et al.,
2017). Thus, it is not clear from previous studies whether observed
increased rates of leaf photosynthesis in intercropped maize were due to
the greater incident radiation, as maize is usually the taller plant in
intercrops, or were due to an increased capacity of the leaves to
photosynthesize because the leaves were accommodated to higher levels
of light, resulting in a greater innate capacity to photosynthesize than
leaves that had grown under more shaded conditions.
Intercrops with maize are often grown as relay intercrops with a C
3
species, whereby the C
3
species is sown and harvested earlier than maize
(Li et al., 2020c). However, in warm climates, such as in Sichuan
province in China (Feng et al., 2020), maize can be the rst-sown spe-
cies. Which species is sown rst greatly affects the competitive re-
lationships and species performance in intercrops (Yu et al., 2016).
Relay intercropping is particularly prevalent in China where species are
usually grown in narrow strips of a few crop rows to facilitate man-
agement as well as interspecic interactions (Li et al., 2020c, 2023). In
relay intercrops in which maize is sown later than its companion species,
maize plants initially experience shading from the early-sown species
(Gou et al., 2017; Zhu et al., 2014). When maize plants overtop the
companion species, and more so after the companion species has been
harvested, maize has an improved access to light, with light penetrating
more deeply into the canopy than in a pure maize stand (Liu et al.,
2018). The yield increase of maize in such relay intercrops compared to
pure stands has been attributed to the enhanced acquisition of light and
soil resources from the strip where the early-sown species is harvested
(Liu et al., 2020; Wang et al., 2023; Zhao et al., 2023). However, the
improvement in light conditions could also result in accommodation of
leaf traits and increased maize leaf photosynthetic capacity after harvest
of a companion species, and an increase in photosynthetic capacity
could also contribute to yield gain. There is little information on the
responses of maize leaves to intercropping in terms of leaf traits and
photosynthesis (Gou et al., 2018) and it is therefore unclear to what
extent such responses may contribute to the yield performance of maize
in relay intercrops.
Previous studies on the response of maize leaf traits to intercropping
have mostly been done either in simultaneous intercrops, in which
maize and a legume were sown and harvested simultaneously (Li et al.,
2019; Liu et al., 2018; Nasar et al., 2020, 2022; Pelech et al., 2022), or in
relay intercrops in which maize was the early-sown species (Feng et al.,
2020; Nasar et al., 2021; Yang et al., 2017). If maize is sown before the
companion species, maize is the dominant species in the intercrop from
the beginning, allowing improved resource capture, e.g., nutrients and
light, which could explain why it would have enhanced leaf traits
compared to sole maize (Feng et al., 2020). In the Netherlands, the
oceanic climate allows maize to be sown only after a C
3
species in a relay
intercrop. A recent study in the Netherlands reported that in
maize-wheat (Triticum aestivum L.) relay intercropping, shading by the
early-sown wheat resulted in increased SLA of maize leaves (Gou et al.,
2018). They also found that intercropped maize had lower leaf N con-
centration (LNC) and lower specic leaf N (SLN; N per unit leaf area)
than sole maize. Further work is required to elucidate how leaf N and
photosynthetic capacity of maize leaves respond to competitive species
interactions and the dynamically changing conditions in relay inter-
cropping. This is relevant for understanding how maize achieves
overyielding in relay intercropping in the Netherlands and to potentially
improve this system.
Leaf traits vary due to species interactions in intercropping that
result in modied access to resources (Evers et al., 2019). Feng et al.
(2020) and Nasar et al. (2021, 2022) found that maize had increased
LNC and SLN when intercropped with soybean (Glycine max L.) or alfalfa
(Medicago sativa L.) compared to sole maize. The increases in leaf N may
be due to N xation by legumes which can x N from the atmosphere,
thus releasing maize from competition for soil N (Brooker et al., 2008;
Fujita et al., 1992). It may also be related to a high N input in inter-
cropping compared to sole maize as many studies were conducted in
China using an additive N input design, i.e., the N input in the intercrop
is the sum of that in the sole crops (Du et al., 2018; Feng et al., 2020; Li
et al., 2020c). Under Dutch growing conditions, additive N input in
intercropping is not acceptable as there are environmental constraints to
N input and N surplus. Hence, to obtain results that are relevant for
European growing conditions, the responses of maize photosynthetic
capacity and leaf traits need to be determined with agronomic practices
that are consistent with European standards for “Good Agricultural
Practices”, i.e., moderate N input (Baghasa, 2008; FAO, 2003).
In strip intercrops, complementarity and competition between spe-
cies are most strongly expressed in the border rows of each strip, and so
are the responses of plant traits (Li et al., 2020a, 2021; Zhu et al., 2015,
2016). Thus, intercropped plants have different traits according to their
position in the strip, which may be true for their leaf photosynthetic
traits as well. This border row effect provides an opportunity to assess
whether effects in intercropping are due to interactions with the
neighboring plants.
In this study, we aim to quantify the extent to which maize leaf
photosynthetic capacity and maize leaf traits are affected by resource
competition with the companion species, under growing conditions that
are relevant for north-west Europe, i.e., with moderate N input and with
maize sown later than the companion species such that maize has a
competitive disadvantage compared to the companion species. We
compare light distribution in the maize canopy in three crop systems:
sole maize, maize-wheat and maize-faba bean (Vicia faba L.) relay strip
intercropping. We compare traits of leaves at different positions in the
maize canopy in the three crop systems and at different times in the
season. We distinguish the responses of maize leaf traits in border rows
and inner rows in the intercrop strips because border and inner plants
experience competitive interactions with different types of neighbors.
We selected wheat and faba bean as two contrasting companion species
for intercropping with maize because previous work in the Netherlands
has shown that wheat-maize relay strip intercropping is a good inter-
cropping system for Dutch growing conditions (Gou et al., 2016). We
anticipated that intercropping maize with a legume could have the
added benet of complementary N use due to biological N xation by
the legume (Bedoussac et al., 2015), allowing a reduction in fertilizer
input, while legumes are needed for generating more plant based protein
for the diversication of food systems and sustainably sourced human
diets (van Zanten et al., 2023).
The study tested three hypotheses: (i) During early growth of maize,
due to shading by wheat and faba bean, leaves of intercropped maize
show shade leaf traits, i.e., larger specic leaf area (SLA), lower specic
leaf N (SLN, leaf N content per unit leaf area), and lower leaf photo-
synthetic capacity than leaves of sole maize. (ii) When maize overtops
the companion species, the upper leaves of intercropped maize experi-
ence better light conditions and thus show sun leaf traits, with lower
SLA, higher SLN, and higher leaf photosynthetic capacity than the upper
leaves of sole maize. After the harvest of the companion species, light
penetrates more deeply in the maize canopy in intercrops. As a result,
leaves in the intercrop from both upper and lower maize canopy show
sun leaf traits when compared to leaves of the same rank in sole maize.
(iii) In maize-wheat intercrops, competition for N between maize and
wheat reduces leaf N concentration (LNC) and SLN of maize compared to
plants in sole maize, whereas LNC and SLN of maize in maize-faba bean
B. Dong et al.
European Journal of Agronomy 155 (2024) 127119
3
intercropping increase compared to plants in sole maize due to com-
plementary N capture between maize and faba bean.
2. Materials and methods
2.1. Experimental design
Measurements were conducted at Droevendaal Experimental Farm,
Wageningen, the Netherlands (51◦59’20’’N, 5◦39’16’’E) in 2018 and
2019 under agronomically realistic growing conditions in the eld
(Wang et al., 2023). We considered three cropping systems: sole maize
(Zea mays L. cv. LG30.223), a relay strip intercrop of maize and spring
faba bean (Vicia faba L. cv. Fanfare), and a relay strip intercrop of maize
and spring wheat (Triticum aestivum L. cv. Nobless). In both intercrops,
species were grown in 1.5 m-wide strips, with three rows of maize or six
rows of wheat or faba bean per strip (Fig. 1). Each species strip had two
border rows (one at each side of the strip) while the other rows are inner
rows.
Maize was grown at a 50 cm row distance. In each wheat or faba
bean strip, the row distance between inner rows was 25 cm, but the
distance between the border rows and the neighboring inner rows was
reduced to 20 cm to allow passage of the tractor wheels (track width
133 cm) without causing damaging the plants in the outer rows of the
strip. We used a replacement design to avoid confounding intercropping
effects with effects of a change in plant density. To obtain a replacement
design (de Wit, 1960), the distance between the border rows of maize
and wheat or faba bean was 25 +17.5 =42.5 cm, where 25 cm was half
the row distance of maize and 17.5 cm was obtained by summing
12.5 cm (half the row distance of wheat or faba bean) and 5 cm (the
distance over which the border rows in a wheat or faba bean strip was
moved “inward” into the strip) (Fig. 1). The relative density (density in
the intercrop relative to the sole crop; van der Werf et al., 2021) for all
species was thus equal to 0.5.
In 2018, wheat was sown on 21 March and harvested on 17 July, faba
bean was sown on 21 March and harvested on 30 July, and maize was
sown on 4 May and harvested on 10 September (Fig. 2). Due to the cool
and wet spring of 2019, wheat was sown on 1 April and harvested on 8
August, faba bean was sown on 1 April and harvested on 14 August, and
maize was sown on 7 May and harvested on 18 September. Maize was
sown at a density of 10 seeds m
−2
and faba bean was sown at a density of
44 seeds m
−2
in both years. The sowing density of wheat was 383 seeds
m
−2
in 2018 and 369 seeds m
−2
in 2019. In 2018, the plot size was 9 m
in east-west ×11 m in north-south directions. Each intercrop plot
comprised six species strips (three of each species). In 2019, the plot size
was 12 m in east-west ×11 m in north-south directions for the sole
maize crop, and 15 m in east-west ×11 m in north-south directions for
the intercrop. Each intercrop plot comprised ten species strips (ve of
each species). The row orientation was approximately north-south in
both years. The experiment had a randomized complete block design
with six replicates in 2018 and four replicates in 2019. Photosynthesis
measurements were made in all replicates.
Soil at the experimental site was sandy with 3.4% organic matter and
a pH of 5.7. While the climate in the Netherlands is oceanic temperate
with mostly cool summers, the summers of the measurements were hot
and dry; hence, sprinkler irrigation was given from June to August, 13
times in 2018 and 9 times in 2019, to avoid drought stress (See Sup-
plementary Figs. S1-S3 for data on daily air temperature, daily photo-
synthetically active radiation, and monthly precipitation). Potassium
was applied in the form of K
2
SO
4
⋅MgSO
4
at a rate of 105 kg K
2
O ha
−1
in
both years. Phosphorus was applied in the form of Ca(H
2
PO
4
)
2
⋅H
2
O at a
rate of 67.5 kg P
2
O
5
ha
−1
in 2018 and 78.75 kg P
2
O
5
ha
−1
in 2019. As
soil P levels were high these rates were based on expected uptake. Po-
tassium and phosphorus were applied homogeneously throughout the
eld before sowing. Mineral N in the 0–30 cm soil layer before sowing
was 22 kg N ha
−1
in 2018 and 12 kg N ha
−1
in 2019. Supplementary N
was supplied in the form of NH
4
NO
3
⋅CaMg (CO
3
)
2
. Total N applied was
20 kg N ha
−1
in faba bean, 125 kg N ha
−1
in wheat, and 170 kg N ha
−1
in maize in both years. N fertilizer in wheat and maize was split into two
doses (Supplementary Table S1). In intercrop plots, fertilizer was
applied within species strips such that plants in the intercrop and sole
crop received the same amount of fertilizer. As the intercrops comprised
50% area of both species, the N input into intercrops per unit intercrop
area was equal to the average of the input in the sole crops of the
component species. Weeds were controlled chemically and manually as
needed. Diseases and pests were managed chemically (Supplementary
Table S1).
2.2. Leaf traits
Photosynthetic capacity of maize leaves was measured to quantify
the effect of interspecic competition during the co-growth period and
when early-sown species in relay intercrops were harvested. Photosyn-
thetic capacity of the youngest full-grown leaf during co-growth with the
companion species was measured at V10 in 2018 and at V6 in 2019
(Fig. 3). In both years, photosynthesis was furthermore measured at
Fig. 1. Schematic illustration of row pattern in sole maize and maize inter-
cropped with spring wheat or spring faba bean. Maize was sown at a 50 cm row
distance in strips comprising three rows. The resulting 1.5 m-wide maize strips
were alternated with 1.5 m-wide strips of wheat or faba bean. Wheat and faba
bean were planted at a row distance of 25 cm, except for the border rows of
each 1.5 m strip, which were moved “inward” into the strip by 5 cm at both
sides to allow space for the wheels of the tractor used for sowing. Each species
in the intercrop had a relative density of 0.5, thus the intercrops followed a
replacement design.
Maize V10 R4
Maize V6 R1 R4
1 Jul 1 Aug 1 Sep 18 Sep21 Mar 1 A pr 1 May 1 Jun
2018
2019
Faba bean
Wheat
Faba bean
Wheat
Fig. 2. Growing periods of wheat, faba bean, and maize in 2018 and 2019.
Measurements on light distribution were made during maize grain-lling (R4)
in 2018, and during six-leaf (V6), silking (R1), and R4 in 2019 (black arrows).
Photosynthesis measurements were made at ten-leaf (V10) and R4 in 2018 and
at V6, R1, and R4 in 2019 (red arrows).
B. Dong et al.
European Journal of Agronomy 155 (2024) 127119
4
maize grain lling (R4), i.e., after harvest of the companion species. In
2018, we made the measurements at R4 on the ear leaf (leaf 10) while in
2019, we measured leaf 14 and a much older leaf (leaf 7) as this older
leaf might show a greater contrast between the sole crop and the
intercrop due to its lower position in the canopy as light is penetrating
more deeply in the maize canopy in the intercrop than in sole maize.
Furthermore, we added an intermediate measurement at maize silking
stage (R1) in 2019, after maize had overtopped its companion species in
both intercrops while these companion species were still present (Fig. 3).
At R1, both a lower canopy leaf (leaf 7) and a upper canopy leaf (leaf 14)
were measured.
We did not make photosynthesis measurements on rainy days or on
days with a maximum temperature above 30 ◦C. In each sole crop plot,
one plant in 2018 and three plants in 2019 were randomly selected. In
each intercrop plot, plants from the western border row of one maize
strip and the adjacent inner maize row were selected. One plant per row
was selected in 2018 and three plants per row in 2019. Measurements
were made in each of the six blocks in 2018 and each of the four blocks
in 2019. The location of the selected plants was at least 1 m away from
the plot edge. Measurements were made on fully developed leaves.
In 2018, we made gas exchange measurements using two portable
photosynthesis systems, the LI-COR 6400XT and LI-6800 (Li-Cor Inc.,
Lincoln, USA). In 2019, we used the LI-COR 6400XT at V6 and R4, and
the LI-COR 6400XT and LI-COR 6800 at R1. When the two LI-CORs were
used simultaneously, all measurements in one block were made with the
same instrument. The LI-COR leaf chamber provided a constant irradi-
ance of 1800 µmol m
−2
s
−1
and a constant CO
2
level of 400
μ
mol mol
−1
.
The photosynthetic rate (A
1800
; µmol m
−2
s
−1
) of maize leaves under this
irradiance is close to the light-saturated rate of photosynthesis, or
photosynthetic capacity (Yin et al., 2011). The adaptation time for each
measurement was 30 min, allowing A
1800
to reach a steady state. Both
A
1800
and stomatal conductance for water (g
sw
; mol m
−2
s
−1
) were then
recorded. Leaf temperature during measurements was maintained at 25
◦C. Leaf-to-air vapour pressure difference was within the range of
1.0–1.5 kPa.
Leaf blades were removed for further analysis after the gas exchange
Fig. 3. Maize development stages and leaf positions for gas exchange measurements in sole maize, the maize-faba bean intercrop, and the maize-wheat intercrop in
2018 and 2019. White arrows represent the approximate positions of the measured leaves in the maize canopy.
B. Dong et al.
European Journal of Agronomy 155 (2024) 127119
5
measurements. Three leaf discs (2.16 cm
2
per disc) were punched
around the position at which the gas exchange measurement was made.
SPAD measurements were made at ten points on each disc, using a SPAD
Meter (SPAD-502, Minolta Camera, Tokyo, Japan) to measure greenness
as a proxy for chlorophyll content. The mean of the ten points was
recorded as the SPAD value for the disc. The fraction of light absorbed by
the leaf, absorptance (Abs), was calculated as one minus transmittance
minus reectance. The transmittance and reectance of each disc was
measured in the spectral range of the light source of the gas exchange
measurement (red: 625–645 nm; blue 455–475 nm), using a Spectrom-
eter (STS-VIS miniature Spectrometer, Ocean Optics, USA). The midrib
was removed and the area of the remaining blade was measured with a
leaf area meter (LI-3100 area meter, Lincoln, USA). The remaining blade
and the three discs were then oven-dried at 70 ◦C until constant weight
to determine dry weight. Thereafter the three discs were ground and the
leaf N concentration (LNC; mg N g
−1
leaf) was analyzed using an
element C/N analyzer (Flash 2000, Thermo Scientic) based on the
Micro-Dumas combustion method. Specic leaf area (SLA; cm
2
leaf g
−1
leaf) was calculated using the area and dry weight of the blade without
the midrib. Specic leaf N (SLN; g N m
−2
leaf) was calculated as LNC
divided by SLA.
2.3. Light distribution
We measured light distribution using a SunScan canopy analysis
system (SunScan SS1, Delta-T Devices Ltd, Cambridge, UK) to determine
relationships between leaf traits and exposure to incoming light. In
2018, these measurements were made at maize R4, and in 2019 they
were made at V6, R1, and R4 (Fig. 2). The measurements were made in
one 1.5 m strip in each sole crop plot, and in one intercrop strip
comprising two 1.5 m species strips in each intercrop plot. The 1 m long
SunScan probe with 64 quantum sensors was placed parallel to the rows
in the canopy (Supplementary Fig. S5), while a Beam Fraction Sensor
(BFS, one quantum sensor) simultaneously recorded the incoming light.
The probe was placed at different heights from the bottom to the top of
the canopy in steps of 25 cm, and from west to east across the rows in
steps of 25 cm, covering the whole strip width and canopy height
(Fig. 4). Measurements were conducted with either clear sky or steady
overcast sky, within two hours from solar noon (around 11:45–15:45)
(for details see in Supplementary Figs. S6 to S9).
As the probe and the reference Beam Fraction Sensor (BFS) gave
slightly different readings, a correction factor is needed to compare them
and determine transmission. Therefore, before light distribution mea-
surements in a plot, the probe and BFS were placed horizontally in
uniform sunlight and three readings were taken to obtain this correction
factor. The corrected probe readings were then used to calculate the
fraction of transmitted photosynthetically active radiation (fPAR) at
different positions in the canopy. fPAR represents the light intensity
detected at any position relative to the light intensity above the canopy.
Visual representations of fPAR distribution in crop canopies were
generated in R version 4.2.1 (R Core Team, 2022), using the “ggplot2”
package (Wickham, 2016).
2.4. Grain yield
Maize was harvested manually at maturity. In each plot, plants from
each row in one strip and at least 1 m away from the edge of the plot
were harvested over a 4 m row segment. The grain yield was determined
after separating the grain from the cobs and drying the grain at 105 ◦C
for 48 hours. The effect of intercropping on grain yield per maize plant
was characterized by overyielding (OY
m
, %) (Li et al., 2011; Wu et al.,
2022; Zhao et al., 2023):
OYm(%) = Yim −0.5Ysm
0.5Ysm
×100
where Y
im
is the grain yield (per unit area of the whole intercrop) of
maize in the intercrop; Y
sm
is the grain yield (per unit area of the sole
crop) of sole maize; 0.5 is the land area ratio of maize in the intercrop in
this study, which was calculated as the strip width of maize (1.5 m)
divided by the width of intercrop strip (3 m; comprises two species
strips); 0.5 ×Y
sm
is the expected yield for maize in the intercrop. As the
sowing density (per m
2
maize strip) was identical in pure stands and the
intercrop, this metric indicates by which percentage the yield per plant
in intercropping exceeds that in the sole crop.
2.5. Statistics
We considered border and inner row maize separately when studying
the responses of maize leaf traits in intercropping. Linear mixed effect
models were used to compare means of ve treatments (sole maize,
maize-faba bean border, maize-faba bean inner, maize-wheat border,
maize-wheat inner) of leaf traits. Comparisons were made for each leaf
position at each growth stage and in each year. In the analyses, treat-
ment was a xed effect and block a random effect. Models were tted
using the function lmer from the “lme4” package (Bates et al., 2015) in R
(R Core Team, 2022). Signicance of the xed effects was determined
with analysis of variance (ANOVA) (P =0.05), using the Anova function
from the “car” package (Fox and Weisberg, 2019). Pairwise comparisons
were conducted using Fisher’s Least Signicant Difference (LSD) in the
“emmeans” package (Lenth, 2021).
To explore relationships between A
1800
and SLA or LNC across
treatments, correlations between A
1800
and SLA, and A
1800
and LNC
were determined for each stage and leaf position per year, using the
combined data from the ve treatments. The R base function lm (R Core
Team, 2022) was used.
3. Results
3.1. Maize leaf traits
Measurements of leaf traits were made in more leaf layers and at
more maize growth stages in 2019 than in 2018. The 2019 data are
Fig. 4. Schematic illustration of the positions of the SunScan probe in the canopy in a maize strip in sole maize and in an intercrop strip in maize-faba bean intercrop
at maize V6 stage in 2019. Every red dot was a measuring point with the probe oriented parallel to the rows. The measurements were made at every 25 cm distance in
the vertical direction, from the ground level to the top of the canopy, and at 25 cm intervals horizontally, from west to east across the rows. The measurement design
in the maize-wheat intercrop was similar to that in the maize-faba bean intercrop (Supplementary Fig. S4).
B. Dong et al.
European Journal of Agronomy 155 (2024) 127119
6
therefore presented rst. The 2018 data are given thereafter to evaluate
consistency.
At V6, A
1800
of the highest leaf, leaf 6, was lower in the border row
maize than in inner row maize or sole maize (Fig. 5 A). This lower A
1800
in the border row was associated with shade leaf traits such as larger SLA
and lower SLN compared to sole maize (Fig. 5 E, G; for substantial
correlation between SLN and SLA see Supplementary Fig. S10). These
shade responses of SLA and SLN were stronger in border row maize in
maize-faba bean than in maize-wheat, which was associated with
comparatively stronger shading of maize in maize-faba bean than in
maize-wheat (see below). On the other hand, border row maize in
maize-wheat had lower LNC than sole maize and both border and inner
row maize in maize-faba bean, indicating that competition for N was
more severe in the intercrop with wheat than in the intercrop with faba
bean (Fig. 5 F).
At R1, leaf 7 was a lower canopy leaf, with seven additional leaves
above it (Fig. 3). Leaf 7 of inner row maize in maize-wheat had higher
A
1800
than the same leaf in sole maize (Fig. 5 A). In intercrops, maize leaf
14 was fully above the canopy of faba bean or wheat. Leaf 14 had higher
A
1800
than leaf 7 in all treatments. No differences in A
1800
of leaf 14 were
found among treatments. Leaf 14 showed sun traits in inner row maize
in maize-wheat, having lower SLA than in sole maize (Fig. 5 E). How-
ever, leaf 14 in border row maize in maize-wheat had lower LNC than
the same leaf in sole maize (Fig. 5 F). Both LNC and SLN of leaf 14 were
reduced in border row maize in maize-faba compared to sole maize
(Fig. 5 F and G). Thus, sun traits occurred in the upper leaves of inter-
cropped maize, but the leaf N was reduced in border row maize in both
intercrops, and A
1800
of leaf 14 was not increased in any intercrop
Fig. 5. Leaf traits of maize in different treatments at different growth stages and leaf positions in 2019. L6: leaf 6; L7: leaf 7; L14: leaf 14; A
1800
: leaf photosynthetic
capacity; g
sw
: stomatal conductance for water; SPAD: a proxy for chlorophyll content; Abs: light absorptance; SLA: specic leaf area; LNC: leaf nitrogen concentration;
SLN: specic leaf nitrogen. Error bars indicate the standard errors of means. In each stage and leaf position, signicance of treatment effects was determined using
ANOVA (* =P <0.05; ns =P >0.05). Details showing the pairwise comparison are presented in Supplementary Table S2.
B. Dong et al.
European Journal of Agronomy 155 (2024) 127119
7
treatment.
At R4, faba bean and wheat had been harvested. In intercrops, the
extra space and resources (i.e., light, water, nutrients) were exclusively
available for maize plants. No differences in A
1800
of maize leaves were
found among treatments (Fig. 5 A), but leaf traits did differ between
treatments. Leaf 14 had lower LNC and SLN in both border and inner
row maize in both intercrops than in sole maize (Fig. 5 F and G; for
substantial correlation between SLN and LNC see Supplementary Fig.
S14). Thus, both leaf N and A
1800
of maize leaves were not increased by
intercropping with faba bean or wheat.
Findings in 2018 were consistent with those in 2019. During the
vegetative phase (V10), the border row maize next to faba bean showed
strong shade responses, having lower A
1800
(Fig. 6 A), larger SLA (Fig. 6
E), and lower SLN (Fig. 6 G) than leaf 10 in sole maize and other
intercrop treatments. At R4, maize A
1800
was not increased in both in-
tercrops after the early-sown species had been harvested.
3.2. Relationships between leaf photosynthetic capacity and the other leaf
traits
We analyzed associations between A
1800
and SLA or LNC to assess
possible causal pathways for effects of intercropping on leaf photosyn-
thetic capacity.
A negative correlation between A
1800
and SLA was found in leaves at
maize V6 and R1 stages in 2019, and at maize V10 stage in 2018
(Fig. 7 A, C, E, and K), indicating shade response (increased SLA; thinner
leaves) as a mechanism for lower leaf photosynthetic capacity during
early maize development in intercrops. A positive correlation between
A
1800
and LNC was found in leaf 14 at maize R1 stage, and in leaves at
maize R4 stage in 2019 and 2018 (Fig. 7 F, H, J, and N), indicating
competition for N between maize and companion species as a possible
mechanism for lower leaf photosynthetic capacity in intercropped maize
during later maize development.
3.3. Distribution of PAR in maize canopy
In 2019, at maize V6 stage, maize leaves in border rows with faba
bean experienced heavier shading than inner row leaves or leaves in
other crop systems (Fig. 8 A to C). At maize R1 stage, upper leaves in the
maize canopy in intercrops were above the wheat or faba bean canopy
and experienced better light conditions than leaves with the same rank
in sole maize (Fig. 8 D to F). At R4 in both years, intercropped maize
showed a deeper penetration of radiation into the canopy compared to
sole maize (Fig. 8 G to K).
3.4. Maize yield
Maize in maize-wheat intercropping produced 27.3% (2018) (P =
0.056) and 16.8% (2019) (P =0.005) more grain yield per plant than
maize in pure stands, while the maize yield was not signicantly
improved in maize-faba bean intercropping (Table 1).
4. Discussion
In this study we tested three hypotheses on the effects of intercrop-
ping on the photosynthetic capacity of maize leaves in relation to spe-
cic leaf area (SLA), leaf N concentration (LNC), and leaf N content per
unit leaf area (SLN). Data are in agreement with the rst hypothesis that
early formed maize leaves respond to shading by an earlier sown com-
panion species, i.e., wheat or faba bean. At maize V6 stage in 2019,
shading from wheat and faba bean resulted in larger SLA and lower SLN
of maize leaf 6 compared to sole maize. Border row maize in both in-
tercrops had decreased A
1800
in leaves with shade traits (Fig. 5 A, E, and
G). These responses were also found at maize V10 stage in 2018: the
shaded leaf 10 of border row maize in the maize-faba bean intercrop had
larger SLA, lower SLN, and lower A
1800
than sole maize (Fig. 6 A, E, and
G).
The second hypothesis posited that intercropped maize exhibits sun
traits in upper leaves formed after maize overtops the companion spe-
cies, and exhibits sun traits in both upper and lower leaves after com-
panion species harvest. This hypothesis was conrmed for leaf 14 in the
inner rows of maize strips when grown with wheat at maize R1 and R4
stages. These leaves had lower SLA than corresponding leaves in sole
maize (Fig. 5 E). However, sun leaf adaptations were not found at other
leaf positions and at other developmental stages in intercropping.
We found evidence supporting the third hypothesis that maize leaves
have lower leaf N when grown with wheat, due to competition for N
between the two cereals, but we did not nd evidence that maize leaves
grown with faba bean have higher leaf N than leaves of sole maize. In
contrast to expectation, LNC and SLN of leaf 14 in border rows of in-
tercrops with faba bean were lower than in sole maize at R1. The same
effect was found for SLN at R4 in 2019 (Fig. 5 F and G). The hypothesis
that complementary N use between maize and faba bean increases leaf N
was thus not conrmed under the conditions of this study.
The results show that competition for light and N with an early-sown
species altered leaf photosynthetic capacity and photosynthesis-related
leaf traits of maize in relay intercrops. The effects were most apparent
in border rows in the intercrop. This is expected because plants in border
rows are directly exposed to resource competition with the companion
Fig. 6. Leaf traits of maize leaf 10 (L10) in different treatments at maize V10 and R4 stages in 2018. In each stage and leaf position, signicance of treatment was
determined using ANOVA with treatment as the xed effect (* =P <0.05; ns =P >0.05). Details showing the pairwise comparison are presented in Supplementary
Table S3.
B. Dong et al.
European Journal of Agronomy 155 (2024) 127119
8
species (Wang et al., 2020). Despite some differences in the experi-
mental protocols between the two years, consistent patterns were
observed: (i) a shading effect on leaf traits during early maize growth in
both intercrops, (ii) evidence for competition for N in maize-wheat
intercropping but (iii) lack of evidence for relaxation of competition
for N in maize-faba bean intercropping, and (iv) no substantial recovery
of maize leaf N and leaf photosynthetic capacity after harvest of the
companion species, despite improved light conditions.
Maize experienced lower light levels during its early growth in relay
intercrops than in a pure maize stand (Fig. 8), conrming earlier studies
in the Netherlands (Gou et al., 2017; Zhu et al., 2014). The maize leaves
accordingly showed shade traits during their early growth, such as a
large SLA Figs. 5 and 6, consistent with earlier work (Gou et al., 2018).
This shade response was associated with a decreased leaf photosynthetic
capacity in border row maize in both intercrops (Fig. 7). Thus, the
shading resulting from interspecic light competition negatively
affected maize leaf photosynthetic capacity. The effects on SLA were
stronger in faba bean-maize than in wheat-maize, indicating that light
competition was stronger with bean than with wheat. This is consistent
with the comparatively tall stature of faba bean plants compared to
wheat and maize (Supplementary Fig. S17) and the shade cast by faba
bean (Fig. 8).
Likewise, in studies on maize-soybean intercropping, shading by the
taller maize plants resulted in thinner leaves and lower leaf photosyn-
thetic capacity of soybean leaves compared to the sole crop (Gong et al.,
2015; Yao et al., 2017). They also found the shaded intercropped
soybean leaves had relatively more chlorophyll b to increase the ca-
pacity for light harvesting. In contrast to soybean (a C
3
species), maize as
a C
4
species is less shade tolerant. A lack of differences in the absorp-
tance values between treatments was found (Fig. 5 D; Fig. 6 D), indi-
cating that the light harvesting of maize leaves was hardly increased
when shaded in intercropping.
In studies on simultaneous intercrops, where maize and compara-
tively low stature species were sown simultaneously, the light condition
of maize was improved, and maize leaf photosynthetic rate measured at
light levels lower than 1800
μ
mol m
−2
s
−1
was higher than in sole maize
(Li et al., 2019; Liu et al., 2018; Nasar et al., 2022). In the subtropical
conditions of Sichuan province, China, where maize is sown before
soybean in a relay intercropping sequence, maize had a higher LNC and
higher leaf photosynthetic rate than sole maize (Feng et al., 2020). The
study of Feng et al. (2020) was done using an experimental design that is
different from ours in several respects: (i) they used an additive design
for species density, maintaining the same number of plants per ha in the
intercrop as in the sole crop, (ii) they used an additive N input strategy in
which the fertilizer input in the intercrop is the sum of the fertilizer
inputs in the two component sole crops, and (iii) the relay sequence is
different. It is therefore not possible to attribute the difference in
photosynthetic response of maize in the Chinese study and our study to
any particular difference in experimental conditions. Results suggest
that the design principles of our study (replacement design and substi-
tutive N fertilizer strategy) are not conducive to maximal photosynthetic
performance of maize; however, the principles used in China,
Fig. 7. Relationships between leaf photosynthetic capacity (A
1800
) and specic leaf area (SLA), and A
1800
and leaf nitrogen concentration (LNC) in 2019 (A to J) and
2018 (K to N). In each stage and leaf position per year, a linear regression was t through the combined data from the ve treatments. Only the lines for regressions
with P <0.05 are presented. Details on coefcients (±SE) and P-values of the regressions are presented in Supplementary Table S4.
B. Dong et al.
European Journal of Agronomy 155 (2024) 127119
9
particularly the high N input in intercropping, may not be acceptable in
Europe because of environmental policies to reduce N leaching. In
addition, after winter in western Europe, C
3
crops are sown before
maize, not the other way around.
In our trials, maize overtopped faba bean only after the appearance
of the tassel (Supplementary Fig. S17). Thus maize plants in intercrops
grew in a shady environment during most of the vegetative growth.
Plants that grow in a shady environment invest relatively more assimi-
lates in leaf area and relatively less in root length (Ryser and Eek, 2000).
This might result in N deciency during later growth. The leaf N of
intercropped maize could thus be reduced, which would then constrain
leaf photosynthetic capacity (Fig. 7). The ndings suggest that inter-
specic light competition during early growth of intercropped maize
may lead to a cascade of physiological effects that result in suppressed N
uptake and ultimately decreases leaf photosynthesis of intercropped
maize during later growth.
In maize-wheat intercropping, N acquisition of intercropped maize is
constrained as wheat is more competitive for N due to its ne root
system and earlier sowing than maize (Gou et al., 2018; Li et al., 2001a;
Liu et al., 2015). In the maize-wheat intercrop, the reduced leaf N re-
ected the effect of N competition with wheat. Despite the N xation
ability of the legume (Bedoussac et al., 2015), light competition with a
vigorous legume, like faba bean in our study, can result in a constrained
access of maize to fertilizer N. The small amount of fertilizer N
(20 kg ha
−1
) applied in the faba bean strip was most likely used up
during its early growth. In contrast to high-input strip intercropping (Li
et al., 2011), in which cereals have extra access to soil N because of N
xation of legumes, the agronomically appropriate low fertilizer input
to the legume in our trials means that maize could in this system not
benet from relaxed competition for N. In the experiments conducted in
China (Liu et al., 2020; Ma et al., 2020), an extra N application was made
at maize tasseling in both relay intercrops and sole crops, to allow
additional N uptake. In our trials, such extra application at tasseling
could have allowed intercropped maize to increase N uptake and thus
better exploit the increased light resource in the late maize growing
season.
The high performance of maize in relay intercropping has been
related to exploitation by maize of the extra light and nutrient resources
that become available after the harvest of the early-sown companion
species (Li et al., 2001b; Wang et al., 2023; Zhao et al., 2023). Maize is
said to “recover” from competition in this type of relay system, a phe-
nomenon referred to as the competition-recovery principle (Zhang and
Li, 2003). Previous studies have indicated that overyielding of in-
tercrops increased with temporal niche differentiation between the two
species (Xu et al., 2020; Yu et al., 2015; Zhao et al., 2023). In the
experiment of Ma et al. (2020), winter wheat was harvested before
maize tasseling, creating a relatively early access for maize to extra re-
sources, increasing leaf photosynthetic rate after the harvest of wheat.
However, a comparison with the study of Ma et al. (2020) is difcult to
make as it is unclear what light levels they used to obtain leaf photo-
synthetic rate. In our trials, both wheat and faba bean were harvested
after the maize tassel appeared. As no recovery is apparent from our
observations it does seem plausible that timing of the release from
competition is important for the resulting leaf traits. This may be further
analyzed in future research.
An early harvest of the companion species in relay intercrops may be
benecial for maize leaf photosynthetic capacity. This can be achieved
by using a winter-sown rather than a spring-sown cereal or legume.
Using a late maturing maize variety if the season length allows could
also be a recommendation to relax intercropped maize from competition
early when maize root system and foliage are still growing. However,
the window of opportunity in the Netherlands is small due to the rela-
tively cool climate, where the temperature sum may not be sufcient for
full maturation of a late maturing maize variety. Use of late maturing
varieties is, however, well possible in warmer climates than the
Netherlands.
We found increased maize yield per plant in the maize-wheat inter-
crop compared to sole maize, but no signicant overyielding of maize in
the maize-faba bean intercrop (Table 1). We also found that leaf
photosynthetic capacity of intercropped maize was negatively affected
by resource competition with both faba bean and wheat. In addition,
maize plants in both intercrops did not have increased leaf area per plant
as compared to the sole crop during the season (Supplementary Fig.
S18). Thus, an enhanced photosynthetic capacity of the intercropped
maize canopy would not be expected. We conclude that changed
Fig. 8. Light distribution in sole maize, maize-faba bean intercropping, and
maize-wheat intercropping at maize V6, R1, and R4 stages in 2019 (A to H), and
at maize R4 stage in 2018 (I to K). The arrows indicate the position of maize
rows. The red lines indicate the position of photosynthesis measurements. The
fraction of photosynthetic active radiation (fPAR) represents the light intensity
detected at each position relative to the light intensity above the canopy. The
fPAR at each position indicates the mean value across blocks. Details showing
the light distribution and weather conditions in each block are presented in
Supplementary Figs. S6 to S9.
Table 1
Overyielding (OY
m
) of maize grain yield and the standard errors (SE) of means in
maize-faba bean and maize-wheat intercropping in 2018 and 2019. P-values
report the outcome of the Student’s t-test to check if the value of OY
m
was
signicantly different from zero (P =0.05).
Year Companion species OY
m
(%) SE P
2018 Faba bean 8.3 9.8 0.434
Wheat 27.3 11.0 0.056
2019 Faba bean 1.7 4.4 0.723
Wheat 16.8 2.3 0.005
B. Dong et al.
European Journal of Agronomy 155 (2024) 127119
10
photosynthetic capacity of maize leaves in intercropping is not a plau-
sible factor for the observed higher maize yields under the conditions of
this study.
In the same experiments Wang et al. (2023) reported that relay in-
tercrops involving maize had advantages in both land productivity and
absolute yield gain compared to sole crops, due to temporal comple-
mentarity between component species in intercrops. In relay inter-
cropping, high productivity of intercrops is in many instances associated
with an increased accumulated light capture compared to the sole crop,
resulting from complementarity in space and time to companion species
(Gou et al., 2017; Yu et al., 2015; Zhang et al., 2008). The observed
overyielding of maize in the maize-wheat intercrop could be explained
by increased light capture when maize overtopped wheat and after the
harvest of wheat, while this may not have been as much the case in the
maize-faba bean intercrop as faba bean was taller than wheat and har-
vested later than wheat. Maize plants intercropped with faba bean
experienced a relatively longer period of shading and had less time to
capture extra light. Thus those maize plants may have had just sufcient
increase in accumulated light capture compared to sole maize to
compensate for the earlier reduction in light capture due to faba bean
shading. Further analysis could be conducted to quantify the accumu-
lated light capture of maize in intercrops to explain the yield perfor-
mance, using models of light interception in heterogenous canopies
(Gou et al., 2017; Li et al., 2021; Zhu et al., 2015).
5. Conclusion
We compared maize leaf traits related to photosynthesis in sole
maize, maize-faba bean and maize-wheat relay strip intercropping in the
Netherlands. Faba bean was taller than wheat causing heavier shading
on maize than wheat did. Accordingly, shade responses were stronger in
maize intercropped with faba bean than with wheat. These shading re-
sponses comprised larger SLA, lower SLN, and lower A
1800
. Intercrop-
ping with wheat or faba bean reduced maize LNC and SLN. Relaxation of
competition after harvest of the early-sown species did not result in
increased leaf N and increased A
1800
in maize; thus we did not observe
the recovery of leaf photosynthetic capacity that we hypothesized. We
conclude that maize leaf photosynthetic capacity was not substantially
improved in relay strip intercropping due to competition for light and
soil N with the earlier sown companion species. Responses of photo-
synthetic capacity of maize leaves did therefore not substantially
contribute to higher maize yields in the studied intercrops, but over-
yielding nevertheless did occur in maize-wheat. The results are related
to intercropping design choices, such as the use of a replacement design
and N fertilization in accordance with each species’ density. The results
suggest that leaf photosynthetic capacity of maize in relay intercropping
could be increased by an earlier relaxation of competition for light and
N, e.g., before maize tasseling, or an extra application of N fertilizer to
maize during the reproductive stage; however, this may be contrary to
the environmental goal of minimizing N leaching after crop harvest.
Hence, further research is needed on the optimization of fertilizer
application in intercrops with maize.
CRediT authorship contribution statement
Bei Dong: Writing – review & editing, Writing – original draft,
Visualization, Methodology, Investigation, Formal analysis, Data cura-
tion, Conceptualization. Zishen Wang: Writing – review & editing,
Methodology, Investigation, Data curation, Conceptualization. Jochem
B. Evers: Writing – review & editing, Supervision, Methodology,
Conceptualization. Tjeerd Jan Stomph: Writing – review & editing,
Supervision, Methodology, Conceptualization. Peter E.L. van der Put-
ten: Resources, Methodology, Investigation. Xinyou Yin: Writing – re-
view & editing, Conceptualization. Jin L. Wang: Writing – review &
editing, Investigation. Timo Sprangers: Writing – review & editing,
Investigation. Xuebing Hang: Investigation. Wopke van der Werf:
Writing – review & editing, Supervision, Project administration, Meth-
odology, Conceptualization.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data availability
The data of this study are available at Data Archiving and Networked
Services (DANS) at https://doi.org/10.17026/dans-z6p-7ane.
Acknowledgement
We gratefully acknowledge the nancial support by the China
Scholarship Council (grant agreement no. 201706350238) and the Eu-
ropean Union’s Horizon 2020 Research and Innovation Programme
(grant agreement no. 727217), project ReMIX: Redesigning European
cropping systems based on species MIXtures, https://www.remix-inte
rcrops.eu. We are grateful to Wageningen Unifarm staff for valuable
help during the experiments. We are grateful to Rutger Vreezen, Maria
Tsolianou, Antoine Galland, Aurelien Gouot, Ren´
ee Beurskens, Zillur
Rahman, Riccardo Missale, and Honghui Ma for their contributions to
the eld experiments.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.eja.2024.127119.
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