Plant Soil (2016) 401:347364

DOI 10.1007/s11104-015-2734-8

REGULAR ARTICLE

Cover crop mixtures including legume produce ecosystem

services of nitrate capture and green manuring: assessment
combining experimentation and modelling
Hlne Tribouillois & Jean-Pierre Cohan & Eric Justes

Received: 9 June 2015 / Accepted: 9 November 2015 / Published online: 1 December 2015
# Springer International Publishing Switzerland 2015

Abstract
Background and aims During the fallow period,
non-legume cover crop species can capture mineral
nitrogen (N) and thus decrease nitrate leaching, whereas
legume cover crop species can provide a green manuring service that increases N availability for the subsequent crop. The aim of our study was to investigate the
ability of bispecific mixtures to simultaneously produce
these two services of N management in relation to their
interspecific interactions.
Methods Three field experiments were conducted at
contrasting sites from summer to autumn to evaluate
25 mixtures and 10 sole crops. We measured biomass,
N acquisition, C:N ratio and soil mineral N. Ecosystem
services were assessed using both experimental data and
simulation model predictions.
Results Overall, prediction of N mineralized from cover
crop residues was significantly higher for mixtures than
for non-legume sole crops. Predictions of nitrate leached
after mixtures did not differ significantly from those
after non-legume sole crops and remained significantly
lower than those under bare soil, especially for mixtures
with turnip rape which benefitted greatly from being in
mixtures.
Responsible Editor: Martin Weih.
H. Tribouillois (*) : E. Justes
INRA, UMR 1248 AGIR, F-31326 Castanet-Tolosan, France
e-mail: helene.tribouillois@toulouse.inra.fr
J.<P. Cohan
ARVALIS-Institut du Vgtal, Station Exprimentale de La
Jaillire, 44370 La Chapelle Saint-Sauveur, France

Conclusions Some of the mixtures provided a choice of

compromises between the two ecosystem services,
which helps define solutions for adapting mixture
choice according to the sites soil and climate characteristics and to fallow period management.
Keywords Catch crop . Complementarity . Green
manure . Intercropping . Legume species . Nitrate
leaching . non-legume species

Introduction
Cover crops (CC) are commonly sown during the fallow
period between two main cash crops to provide a wide
range of ecosystem services within agro-ecosystems.
CC can provide ecosystem services concerning nitrogen
(N) management in annual crop rotations (Snapp et al.
2005). They can be particularly effective at acquiring
soil mineral N (SMN) and thus reducing nitrate (NO3)
leaching and water pollution (the Bcatch-crop effect^).
They can also increase soil N availability for next crop
once their residues mineralize, allowing less N fertilizer
to be applied and producing a Bgreen manure effect^
(Thorup-Kristensen et al. 2003; Justes et al. 2012).
However, the integrative effect of both nitrate leaching
mitigation by CC plants and N release from CC residues
can lead to contrasted situations according to climatic
conditions occurring during the fallow period. A positive impact of CC allows higher SMN available for the
next cash crop than bare soil, in particular when the
drainage and nitrate leaching are high. At the opposite,

348

a negative impact of CC induces a lower SMN availability for the next cash crop in comparison with bare
soil, due to pre-emptive competition of CC not balanced
by a surplus of N mineralization from CC, in particular
in dry winter. A wide range of plant species can be used
as CC, including those from botanical families such as
Fabaceae, Poaceae, Brassicaceae or Hydrophyllaceae.
Although species from these families can provide both
N-management ecosystem services, legume species
(Fabaceae) would mostly produce a green manure effect. These species can maximize N acquisition by fixing atmospheric N2 through symbioses with N-fixing
rhizobacteria, accumulating a large amount of N and
increasing soil N availability for the subsequent cash
crop after their incorporation (Touchton et al. 1984;
Peoples et al. 1995; Tonitto et al. 2006). Species from
non-legume families would mostly take up SMN, especially nitrate, decreasing N leaching (Meisinger et al.
1991; Constantin et al. 2011; Thomsen and Hansen
2014). Growing CC species in intercropped mixtures
(IC) composed of a legume and a non-legume species
may maximize the benefits of each species due to the
principle of niche complementarity (Jensen 1996b). The
N2 fixation and nutrient cycling could provide additional ecosystem services compared to sole crop (SC) CC
(Ranells and Wagger 1997; Kuo and Sainju 1998;
Wortman et al. 2012a). Mixing species with good complementarity in growth and N acquisition can reduce the
C:N ratio in order to promote N release for the next crop
and avoid the N immobilization for the subsequent cash
crop commonly observed with non-legume sole CC
such as those in the Poaceae or Brassicaceae families
(Quemada and Cabrera 1995; Tosti et al. 2012). Most
studies of intercropping focus on legume-cereal mixtures as main crops. The cereal species in these mixtures
tended to be stronger competitors and took up SMN
(Hauggaard-Nielsen et al. 2001), while the legumes
fixed more N than they would have as a SC (Jensen
1996b). Further studies investigated legume and nonlegume mixtures as CC and illustrated that species in
mixtures tend to increase productivity or the production
of ecosystem services (Ranells and Wagger 1997; Kuo
and Sainju 1998; Mller et al. 2008; Kramberger et al.
2013; Tosti et al. 2014; Summers et al. 2014). The Land
Equivalent Ratio (LER) was developed to analyze land
use efficiency and quantify the potential advantage of
mixed cropping in comparison to sole cropping (Willey
1979) and has been used to analyze performances of
multi-species main crops, such as durum wheat and

Plant Soil (2016) 401:347364

winter pea (Bedoussac and Justes 2011). Only a few

studies have used the LER to evaluate interactions in CC
mixtures (Wortman et al. 2012b; Schipanski and
Drinkwater 2012; Smith et al. 2014). To our knowledge,
no available study in the literature uses the LER to
evaluate performances of multiple bispecific CC
mixtures.
The purpose of this study was twofold: i) evaluate the
abilities of multiple bispecific (one legume and one
non-legume) CC mixtures to provide ecosystem services related to N management compared to the abilities
of SC and bare soil and ii) analyze magnitudes of
interspecific interactions and mixtures abilities to simultaneously produce both ecosystem services for N
management. To this end, we tested the hypothesis that
some bispecific legume and non-legume CC mixtures
can simultaneously and effectively produce both ecosystem services: i) acquiring N from soil and reducing
nitrate leaching as effectively as non-legume SC and ii)
acquiring more N thanks to N2 fixation, reducing the
C:N ratio and thus releasing more N for the next crop as
produced by legume SC. We evaluated performances of
CC mixtures by combining data from field experiments
conducted from late summer to late autumn and simulations of the STICS soil-crop model to estimate both
ecosystem services of nitrate capture and green
manuring.

Materials and methods

Experimental sites and design
Three experiments were conducted from August to
November 2012 at three experimental sites in France:
i) the Institut National de la Recherche Agronomique
(INRA) site at Auzeville-Tolosane, southwestern France
(4331 N, 130 E); ii) the Arvalis-Institut du vgtal
sites at Bignan, western France (4752 N, 246 W) and
iii) at Lyon, eastern France (4543 N, 54 E). Site
descriptions and growth conditions are detailed in
Table 1. The Bignan site had higher levels of soil organic matter and initial SMN available at the CC sowing
date than the two other sites. The main crop before CC
sowing was faba bean (Vicia faba) at Auzeville, barley
at Bignan and wheat at Lyon. The Lyon site was very
pebbly, with approximately 30 % pebbles from 0 to
30 cm in depth, 50 % from 30 to 60 cm and up to
70 % from 60 to 90 cm (deep alluvial gravel soil type)

Plant Soil (2016) 401:347364

349

Table 1 Summary of management and sampling dates, soil and climate at the three experimental sites. Climate variables are sums or means
of the growing season data from the sowing date to the cover crop destruction (biomass harvest date)
Site description and climate

Auzeville

Bignan

Lyon

Sowing date

16 Aug. 2012

17 Aug. 2012

01 Aug. 2012

Biomass harvest date

26 Oct. 2012

13 Nov. 2012

21 Nov. 2012

Soil texture

Clay loam

Silt loam

Loam

Soil mineral nitrogen at sowing (kg N ha1)a

53

112

31

Soil organic matter (030 cm) (g kg1)

14

34

16

Cumulative irrigation (mm)

60

Cumulative rainfall (mm)

80

323

331

Cumulative evapotranspiration (mm)

237

159

343

Daily mean temperature (C)

19.1

13.5

18.4

Daily mean global radiation (J cm2)

1430

1031

1187

Soil mineral nitrogen (N) measured at 090 cm for Bignan and Lyon and 0120 cm for Auzeville

and was thus highly sensitive to nitrate leaching. The

three sites represented a wide range of soil and climate
conditions, which was necessary to test our hypothesis.
The experimental design was a complete randomized
plan replicated with three blocks for the INRA site and
two blocks for the Arvalis sites due to limited technical
support. Surface area of the elementary plot for each
treatment ranged from 14 m2 (Auzeville) to 25 m2
(Bignan and Lyon). The CC treatments were i) ten SC
(five legumes and five non-legumes); ii) 25 bispecific
mixtures composed of one legume and one non-legume,
corresponding to an orthogonal bispecific crossing of
the five legumes x five non-legume species; and iii) a
bare soil, without any CC or volunteers, as the control.
Species were chosen to represent botanical family diversity and shoot/root architectures and to have a sufficiently rapid growth rate for use as a CC for short
growth periods in autumn. We chose non-legume species with a height compatible with legume species to
limit light competition. Ten species were selected: i) five
legumes: crimson clover (Trifolium incarnatum, cv.
Cegalo), purple vetch (Vicia benghalensis, cv. Bingo),
wild lentil (Lens nigricans, cv. Fentille), forage pea
(Pisum sativum, cv. Assas) and faba bean (cv. Laura)
and ii) five non-legumes: turnip rape (Brassica rapa, cv.
Avalon), foxtail millet (Setaria italica, cv. Tardivo),
bristle oat (Avena strigosa, cv. Panache), Italian ryegrass
(Lolium multiflorum, cv. Suxyl) and phacelia (Phacelia
tanacetifolia, cv. Stala). Species in SC were sown at the
densities recommended for CC use and to develop homogeneous dense plant covers with rapid growth and
soil cover. In mixtures, sowing densities were half of the

corresponding SC density of each species (50 %:50 % of

each SC), to create a substitutive design. Seeds of both
species were mixed in the row. No fertilizer was added
during the experiments. At the Auzeville site, irrigation
was provided after sowing to ensure plant emergence
because the soil was dry and no rain was forecasted for
two weeks. No irrigation was performed after this stage
at this site, and none was performed at the Bignan and
Lyon sites. For all sites, superficial tillage was carried
out before CC sowing. Weed control was performed
manually from the sowing on identified sampling plots
at Auzeville. For other sites no weed control on CC plots
was performed, however weeds biomass and N acquisition were measured and represented less than 10 % of
total biomass. Weed control was strictly performed on
bare soils.
Measurements and calculations
Samples of CC aerial biomass were harvested at the soil
surface from an area of ca. 1 m2 for each replicate.
Sampling occurred in autumn 2012 on 26 October for
Auzeville, 13 November for Bignan and 21 November
for Lyon (Table 1). These days corresponded to potential
days of CC destruction and incorporation into the soil
before winter crop sowing. The aerial biomass of each
species was separated, dried at 80 C for 48 h and
weighed. To account for differences in the duration
of the growing period, we estimated crop growth
rate (CGR) in kg ha 1 DD 1 , where DD is
degree-days using a single base temperature of
0 C for all species, calculated from the date of

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Plant Soil (2016) 401:347364

sowing until the date of sampling (Tsampling), using

DM sampling
t h e f o l l o w i n g e q u a t i o n : CGR T sampling
(see
Tribouillois et al. 2015 for details), where DMsampling
is shoot dry matter (DM) (kg ha1) at Tsampling. The sum
of degree-days was 1380 DD at Auzeville, 1200 DD at
Bignan and 1870 DD at Lyon, corresponding to 72, 89
and 114 days after sowing, respectively. After weighing
shoot DM, a subsample of each treatment was ground to
measure total N and total C concentrations by elemental
analyses based on the Dumas method (Elementar
MicroVario Cube, Germany) and 15N concentration
with a stable isotope ratio mass spectrometer in continuous flow (Isoprime 100, UK). To estimate the total
(shoot + root) amount of N acquired by CC, we also
harvested root biomass to a depth of 25 cm for SC
treatments and used the same N analysis methods that
had been used for shoots. The root:shoot ratio was
assumed to be constant for a given species, whether in
SC or in mixture, to estimate the total amount of N
acquired for all treatments.
For legumes, we calculated the amount of N2 fixed
by estimating the percentage of plant N derived from the
atmosphere (%Ndfa) using the 15N natural abundance
(15N) method (Amarger et al. 1979; Unkovich et al.
2008). It was calculated using the following equation:
%Ndfa 100*

15 Nref  15 Nlegume
15 Nref  fix

where fix is the isotopic fractionation factor for legume

N2 fixation. The fix was considered equal to 0.78 for
faba bean (Peoples et al. 2002), 1.55 for forage pea
(Schipanski and Drinkwater 2012), 1.18 for wild lentil,
0.67 for crimson clover (Gebhard 2012) and 0.79 for
purple vetch (Unkovich et al. 2008). The values of
15Nref for mixtures were those of the associated
non-legume species, and for legume SC, they were the
mean of nearby non-legume species. To estimate the
amount of SMN taken up by legumes, we subtracted
the amount of N that each fixed from the total amount of
N accumulated in its total biomass.
Soil samples were taken at depths up to 90 cm
(Bignan and Lyon) and 120 cm (Auzeville) with a
hydraulic core drill shortly after biomass sampling. Six
soil cores were sampled on each plot and each replicate
to account for spatial variability. The six cores were then
pooled to determine water content and mineral N (nitrate
and ammonium). Soil mineral content was measured
with KCl extraction through colorimetric reactions in a

continuous flow autoanalyzer (Skylar 51,000, Skalar

analytic, Erkelenz, Germany).
Estimating ecosystem services with the STICS model
We used the STICS soil-crop model to estimate green
manure and catch crop services produced by the SC and
intercropped CC. STICS is a dynamic model that simulates C, N and water cycles with a daily time step
according to soil and climate characteristics and agricultural practices (Brisson et al. 1998, 2002, 2003, 2008). It
was parameterized for and evaluated as satisfactory in
predicting monospecific CC residue decomposition by
Justes et al. (2009). It is also accurate at predicting
nitrate leaching under monospecific CC (Constantin
et al. 2012; Tribouillois 2014; Coucheney et al. 2015).
We performed the simulations in two steps for targeting
two different objectives (Fig. 1). First, we ran the model
from sowing to the date of CC harvest and destruction
(at the date of biomass and soil measurements) in order
to evaluate if there was some drainage and nitrate
leaching during CC growth in comparison to bare soil.
This first step allowed characterizing the water and N
balances during the growing period for the three sites in
order to relevantly interpret the results and propose more
generic conclusions. According to the capacity of the
STICS soil-crop model, we simulated the growing period of CC only for legume SC, non-legume SC and the
bare soil, and not for mixtures. For this, the STICS
model has been already fully optimized and validated
for three CC species (mustard, ryegrass and vetch)
(Dorsainvil 2002; Constantin et al. 2015). Based on
these species we optimized, for the other CC species
studied, some specific parameters about plant growth
and N acquisition using the JavaStics optimizer based
on the simplex algorithm (Guillaume et al. 2011). In this
way, the parameters were optimized based on variables
measured in the experimental fields described above
(biomass, N acquired, amount of N in soil profile and
N fixed for legumes) in order to have the best simulation
of correlated variables of interest (e.g. nitrate leaching).
Some other parameters were directly measured in fields
such as the duration of the various stages, frost sensibility and root development rate in depth. The objective of
this first step was to obtain a simulation of the various
CC species in order to simulate nitrate leaching as well
as possible during the CC growing period. Even if
STICS is able to simulate intercropping (Brisson et al.
2004), we were not able to accurately optimize and

Plant Soil (2016) 401:347364

351

Fig. 1 Schematic representation

of the different simulations
carried out with the STICS model
in order to evaluate the ecosystem
services related to N management
produced by cover crops (CC)

predict the growth and N acquisition of CC species in

mixtures. However, in the second step, no new parameterization was needed for simulating the effect of CC
residues incorporation on soil N processes thanks to a
previous parameterization done by Justes et al. (2009)
for a wide range of CC species having a C:N ratio
between 9.5 and 34.0. Then we began simulations for
all treatments at the CC destruction date (biomass
harvest) by using measured data for initiating the
STICS model. The simulation aimed at evaluating
the catch crop and green manure services as cumulative amounts of i) nitrate leached until the end of the
drainage period of the following calendar year (31
March) at 120 cm depth for Auzeville and at 90 cm
depth for Bignan and Lyon sites and ii) N mineralized
from CC residues after mechanical soil incorporation
until the period that N is required by the next cash
crop (31 May). In order to evaluate the integrative
effect of both services, we also estimated the amount
of SMN simulated at 31 March -which corresponds to
the SMN available for next cash crop at sowing
period- as a result of the initial SMN at the date of
CC incorporation and various concomitant dynamic
processes (i.e.: N mineralization from soil organic-N
and CC residues, and nitrate leaching). We simulated
the three experimental sites with their specific soil
characteristics, initial soil stocks (water, NO3 and
NH4+ contents in each 30-cm soil layer), and weather
data recorded during the experiments. To simulate the
N mineralized from CC residues, we used the measurements of CC biomass and their C:N ratios as
model input data to be as precise as possible in
assessing the two N-related ecosystem services for
each treatment.

Evaluating interactions in mixtures

The efficiency for the use of abiotic resources of species
in bispecific mixture was compared to SC by using the
concept of LER. The LER is defined as the area of land
required by SC to achieve the same aboveground
biomass that they attain in a mixture (Willey
1979). We calculated the LER for N acquired by
a CC mixture as the sum of the partial LER
(LERp) values for both legume and non-legume speNacqIC
cies: LERp Nacq
and LER = LERplegume + LERpnon SC

legume (see Bedoussac and Justes 2011 for details), where

NacqIC is the N acquired by a species (legume or

non-legume) in the mixture and NacqSC is N acquired
by the same species as a SC. When a mixture reaches an
LER > 1, it indicates that its species use resources more
efficiently than when they are in SC. Conversely, when
LER < 1, mixtures species do not use resources (competition stronger than complementarity or facilitation) as
well as they do in SC. LERp indicates the degree to
which each species benefits from being in a mixture. In
our case, LERp is compared to a threshold of 0.5,
because each species in a mixture was sown at half of
its SC density. As a consequence, the LERp indicates
whether each species was positively (LERp > 0.5) or
negatively (LERp < 0.5) affected by interactions occurring in the mixture. LERp and LER were calculated for
each replicate.
Statistical analyses
Data for CC performances were evaluated with analysis
of variance (ANOVA), which considered the treatment

352

as a factor to explain each variable. If significant treatment differences were detected, we performed a
posteriori Newman-Keuls tests with a level of significance of P < 0.05. Statistical tests were performed
separately for each site to detect site-specific effects.
Statistical analyses were performed with
STATGRAPHICS Centurion XV software (version
15.2.06).

Plant Soil (2016) 401:347364

with turnip rape had significantly lower SMN than

those with other non-legume species. For other
sites, no significant difference was found among
non-legume species in mixtures on the date of CC
destruction, partly due to drainage and leaching
(Fig. 4b-c), which masked differences among treatments. The legume species in the mixtures had no
significant impact on SMN on the destruction date,
except at Lyon, where forage pea led to significantly higher SMN.

Results
Interspecific interactions in mixtures
N acquisition of cover crop mixtures and soil mineral N
Overall, mixtures at all sites tended to acquire more total
N than non-legume SC (Fig. 2, Table 2). At both sites of
Auzeville and Lyon, mixtures with turnip rape, bristle
oat, foxtail millet and Italian ryegrass (mean of each
non-legume intercropped with the five legumes) acquired significantly more N than their corresponding
SC due to N2 fixed by legumes, as expected. Mixtures
with turnip rape and foxtail millet acquired significantly
less N than mixtures with other non-legume species
(Fig. 2a, e). Conversely, at Bignan, mixtures with turnip
rape acquired the most N (Fig. 2c, d). Mixtures with
faba bean fixed significantly more N2 than mixtures
with forage pea or purple vetch, which fixed significantly more than mixtures with crimson clover or wild lentil
(mean of each legume intercropped with the five nonlegumes). However, at Lyon, mixtures with crimson
clover acquired the most N from N2 fixation. The results
indicate that mixtures tended to acquire as much or
slightly less SMN than non-legume SC (Fig. 2;
Table 2). Legume SC also acquired significant amounts
of SMN, especially at Lyon, where faba bean, forage pea
and purple vetch acquired approximately 80 kg ha1 of
SMN.
At all sites, SMN measured on the date of CC
destruction was significantly lower under all CC
treatments than under bare soil (Fig. 3; Table 2).
However, significant differences between mixtures
and SC were found particularly at Auzeville due to
low rainfall and no drainage or leaching on this
date (Fig. 4a). Thus, the residual SMN under mixtures was similar to those under non-legume SC,
which were lower than those under legume SC.
This was particularly true for wild lentil, forage
pea and purple vetch, which had significantly lower SMN than bare soil. At Auzeville, mixtures

At Auzeville and Lyon, almost all CC mixtures had

LER >1, whereas at Bignan only 11 of 25 mixtures had LER > 1 (Fig. 5). At Auzeville and
Lyon, mixtures had high mean LER (1.26 and
1.38, respectively), which were significantly higher
than those at Bignan (mean LER = 0.96).
Variability in species behaviors in mixtures produced a wide range of LERp values for legumes
and non-legumes, indicating great variability in
species responses according to interspecific interactions, which probably varied according to resource availability. Most mixtures (20 of 25) at
the three sites benefitted the non-legume species
(LERp > 0.5). However, only a few mixtures
benefitted legume species at Auzeville and
Bignan, whereas at Lyon, legume species were
highly advantaged in most mixtures in comparison
to sole crops. Finally, 2, 3 and 14 mixtures at
Bignan, Auzeville and Lyon, respectively, experienced
an increase in N acquisition in mixtures for both species
(LERp of legume and non-legume >0.5). Turnip rape
at the three sites was the most favored species in
mixtures (LERp > 0.5) but was also often had
LER close to or higher than 1, indicating a better
performance in mixtures than in SC. Bristle oat at
Auzeville and purple vetch at Lyon also had an
LERp close to 1. Conversely, at Auzeville,
phacelia was strongly disadvantaged in mixtures,
with a low LERp of 0.30. At Bignan, only turnip
rape LERp (1.00) was significantly higher than
those of other non-legume species (mean
LERp = 0.60). Concerning the effect of species
in mixtures on the LERp of a given species, we
highlight turnip rape and bristle oat, which decreased the LERp of the associated legume at
Auzeville and Bignan, whereas foxtail millet and

Plant Soil (2016) 401:347364

353

Fig. 2 N acquired via N uptake and N2 fixation by non-legume

(left) and legume (right) species in mixtures (IC) and in sole crops
(SC) at three experimental sites. Data of non-legumes in mixture
correspond to the mean of each non-legume with the different
intercropped legumes. At the opposite, data of legume in mixture

correspond to the mean of each legume with the different

intercropped non-legumes. The symbols B***^, B**^ and B*^
indicate significant differences at P < 0.001, P < 0.01 and
P < 0.05, respectively

phacelia decreased the LERp of the associated

legume at Lyon. Italian ryegrass at the three sites
was the non-legume species that induced the least
competition, since it induced the highest LERp of
the associated legume, probably due to its relatively lower growth rate after emergence than those of
other species.

Ecosystem services related to N

No significant difference was measured in the
mean CGR of mixtures and SC at Auzeville and
Bignan, but at both sites, legume SC tended to
produce less biomass. At Lyon, mixtures and legume SC produced significantly more biomass

2.3 0.5 a

2.4 0.5 a

SC Leg

IC

Bare soil

Bare soil

1.9 0.6 b

2.8 1.0

SC NL

2.2 1.0

IC

Bare soil

SC Leg

1.6 0.4

IC

3.1 1.0

1.4 0.4

SC Leg

SC NL

1.5 0.4

SC NL

118 35 a

139 47 a

69 28 b

87 30

87 48

79 20

67 15 b

78 21 a

47 19 c

Total N
acquired
(kg ha1)

48 29 b

83 57 a

0c

18 19 b

45 35 a

0c

20 13 b

46 15 a

0c

N2 fixed
(kg ha1)

71 24

64 33

69 28

70 25 a

42 16 b

79 20 a

48 12 a

31 11 b

47 19 a

Mineral N
catched from
soil (kg ha1)

19 6 b

14 2 c

24 6 a

16 3 b

12 2 c

19 3 a

15 4 b

11 1 c

20 6 a

C:N ratio

65 18 a

31 16 b

37 16 b

22 8 b

96 2 a

66 18 b

68 21 b

66 17 b

89 21 a

31 12 c

46 12 b

28 9 c

Mineral N at
CC destruction
in soil 090 cm
(kg ha1)

32 a

6* b

82b

35b

48

32*

39 10

32 16

57 15 a

30 15 bc

43 16 ab

15 5 c

85 6 a

63 10 bc

72 16 b

56 8 c

48 11 a

18 9 c

28 4 b

15 6 c

leached after
CC destruction
(kg ha1)

leached from
sowing to CC
destruction
(kg ha1)
0

Simulated NO3

Simulated NO3

24 21 b

46 19 a

27c

22 9 b

36 14 a

13 5 c

22 8 b

34 7 a

11 13 c

Simulated mineralized
N from CC residues
(kg ha1)

28 5 bc

35 12 b

48 11 a

20 5 c

45 0 c

53 3 ab

57 7 a

50 2 b

69 12 a

56 9 b

73 8 a

46 9 c

Simulated soil
mineral N in soil
profile for next
cash crop (kg ha1)

/ means no corresponding data; * means it was estimated as a proportion of Mineral N at CC destruction. Letters after values indicate treatments with significant differences at P < 0.05

Lyon

Bignan

Auzeville

Crop growth
rate (CGR)
(kg ha1 DD1)

Table 2 Cover crop (CC) performances measured at the date of CC destruction (six first columns) and simulated with the STICS model (last four columns) at three experimental sites.
Values of measured or simulated variables for SC NL correspond to the mean ( standard deviation) of the five non-legume (NL) species in sole crops (SC), those of SC Leg correspond to
the mean ( standard deviation) of the five legume (Leg) species in sole crops, and those of IC correspond to the mean ( standard deviation) of the 25 different intercropped mixtures (IC).
DD is degree day (basis 0 C)

354
Plant Soil (2016) 401:347364

Plant Soil (2016) 401:347364

355

Fig. 3 Amount of soil mineral N in soil layers under mixtures (IC)

and sole crops (SC) measured on the date of cover crop destruction
and under bare soil for the three experimental sites: a Auzeville, b
Bignan and c Lyon. Data of non-legumes in mixture correspond to
the mean of each non-legume with the different intercropped

legumes. At the opposite, data of legume in mixture correspond

to the mean of each legume with the different intercropped nonlegumes. The symbols B**^ and B*^ represent significant differences in soil mineral N in the total soil profile (sum of all layers)
between IC and SC at P < 0.05 and P < 0.10, respectively

than non-legume SC (Table 2). In addition, mixtures had significantly lower C:N ratios than

non-legume SC but significantly higher C:N ratios

than legume SC. As a consequence and as

356

Fig. 4 Dynamic representation of cumulative nitrate leaching and

water drainage simulated with STICS model before and after cover
crop destruction under bare soil, Italian ryegrass (example of non-

Plant Soil (2016) 401:347364

legume sole crop) and purple vetch (example of legume sole crop).
The monthly rainfall measured at the three experimental sites
explains the drainage

Plant Soil (2016) 401:347364

expected, the STICS model predicted a significantly higher amount of N mineralized from CC residues after incorporation for mixtures than for nonlegume SC but it remained significantly lower than
those for legume SC (Table 2) due to lower N
acquisitions and higher C:N ratios.

Fig. 5 Partial Land Equivalent

Ratios (LERp) based on N
acquired by legumes versus
LERp of non-legumes in mixtures
at the three experimental sites: a
Auzeville, b Bignan and c Lyon.
Symbol colors correspond to the
non-legume species in mixtures,
while symbols correspond to the
legume species in mixtures.
Vertical and horizontal dashed
lines indicate LERp of 0.5, which
represents neutral interactions
between species. The diagonal
dashed line indicates a total LER
of 1, at which a mixture is equal to
its sole crops

357

STICS predictions of nitrate leaching in SC from

sowing to the date of CC destruction indicated that no
leaching occurred regardless of the treatment at
Auzeville, even under bare soil. Conversely, nitrate
leaching was predicted to occur under bare soil and
CC treatments at Bignan (high predicted drainage of

358

approximately 200 mm under bare soil and SC, Fig. 4b)

and at Lyon (high predicted drainage of 153 mm under
bare soil and 90 mm under SC, Fig. 4c); significant
differences were found at Lyon between CC treatments
and bare soil (Table 2). At the three sites, the predicted
amount of nitrate leached after incorporation was not
significantly different under CC mixtures or nonlegume SC and remained significantly lower than that
under bare soil, indicating the ability of CC mixtures to
strongly reduce nitrate leaching despite the early date of
incorporation in autumn (Table 2).
At Auzeville, where no leaching occurred during the
CC growth period, mixtures with turnip rape had the
lowest predicted nitrate leaching after CC destruction
(7 kg N ha1), which was almost equal to that induced
by turnip rape in SC (Fig. 6). Mixtures with Italian
ryegrass intercropped with the five legumes induced
more predicted nitrate leaching (23 kg N ha1) than
those with other non-legume species. The simulations
also indicated that legume SC would decrease nitrate
leaching significantly more than bare soil. A significant
relationship was found at Auzeville between measured
SMN on the date of CC destruction and the predicted
nitrate leached after destruction and incorporation
(Fig. 7) illustrating the importance and effectiveness of
CC in reducing residual SMN in the soil before drainage
and leaching begin in autumn.
Finally, for the three sites, the predicted amount
of SMN which would be available for the next
cash crop after CC mixtures would be comprised
between the values of non-legume SC (the lowest)

Fig. 6 Simulated amount of

NO3 leached after cover crop
destruction at (left) non-legume
and (middle) legume treatments
and (right) under bare soil at the
Auzeville site. Data of nonlegumes in mixture correspond to
the mean of each non-legume
with the different intercropped
legumes. At the opposite, data of
legume in mixture correspond to
the mean of each legume with the
different intercropped nonlegumes. The symbol B*^
represents a significant difference
in nitrate leaching under mixtures
and sole crops at P < 0.05

Plant Soil (2016) 401:347364

Fig. 7 Correlation between simulated amounts of NO3 leached

after cover crop (CC) destruction and measured amounts of soil
mineral N from 0 to 120 cm under CC mixtures, sole crops (SC)
and bare soil on the CC destruction date at Auzeville

and of legume SC (the highest) (Table 2). The

simulated SMN after CC mixtures was also significantly higher than after bare soil at Bignan, where
drainage was the highest during winter (Fig. 4b).
At the opposite, SMN was lower after CC than for
bare soil at Auzeville, where drainage was very
low during winter (Fig. 4a).
Catch crop and green manure services produced by CC
mixtures
We calculated the degree to which mixtures produced
the two ecosystem services by comparing mixtures to

Plant Soil (2016) 401:347364

SC, which would serve as references for optimal

provision of services. We assumed that the catch crop
reference level (100 % achievement) corresponded to
the mean service provided by non-legume SC, while
the green manure reference level corresponded to the
mean service provided by legume SC. No mixture
produced both services with 100 % achievement
(Fig. 8); nevertheless, the diversity of mixtures
yielded a variety of degrees of ecosystem service
achievement. The most efficient mixtures differed
among sites. Some mixtures produced both ecosystem
services with a good level of achievement (i.e. intermediate between the two SC references), for example,
those with i) bristle oat or lentil at Auzeville, ii)
Fig. 8 Performances of mixtures
in providing catch crop and green
manure services for the three
experimental sites: a Auzeville,
b Bignan, c Lyon. The catch crop
service was defined as the degree
to which mixtures achieve the
mean amount of nitrate leached
after cover crop destruction of
non-legume sole crops (white
dots). The green manure
service was defined as the degree
to which mixtures achieve the
mean amount of N mineralized
from cover crop residues of
legume sole crops (black dots).
Red dots correspond to the
target for mixtures to produce
100 % of both services. The black
line between the white and black
dots represents the potential
compromise between production
of the two services by bispecific
legume and non-legume mixtures

359

phacelia or faba bean at Bignan, and iii) turnip rape

or crimson clover at Lyon. At Bignan, all mixtures
achieved at least 71 % of the catch crop service. At
Lyon, high variability was observed among mixtures,
with the mixture of foxtail millet and lentil producing
a high catch crop service (155 % of non-legume SC)
but a negative green manure effect (27 % of legume
SC). However, some mixtures at Lyon such as ryegrass and forage pea produced a high green manure
service (115 %) and a low catch crop service (26 %).
At Auzeville, all mixtures with turnip rape had high
efficiency in producing a catch crop service
(>200 %), indicating that this cruciferous plant grew
better in a mixture than in a SC.

360

Discussion
Green manure service of CC mixtures
Our results confirmed that bispecific legume/nonlegume CC mixtures tended to be more efficient in
providing a green manure effect than non-legume SC
and, as a consequence, would improve growing conditions for the subsequent crop by increasing N availability (Snapp et al. 2005). This is supported by increased N
accumulation in the plants, and especially by additional
substantial amounts of atmospheric N2 fixed by the
legumes and a reduction in the C:N ratio, which led to
more N mineralized from mixture residues and available
for the subsequent crop. These results are consistent
with those of Tosti et al. (2012) for a barley-hairy vetch
mixture used as a CC to provide green manure. At the
three sites, despite the effect of interspecific competition, the fact that legumes in mixtures fixed approximately half of the amount fixed by legumes in SC, can
be explained by a higher rate of N2 fixation due to a
lower nitrate content in the upper layers of the soil.
Moreover, the green manure effect combined with the
decrease of N leaching in CC mixtures was efficient to
limit the pre-emptive competition for N which can be
induced by non-legume SC in comparison to bare soil.
The CC mixtures allowed then increasing SMN available for the next crop in comparison to non-legume SC.
Cover crop mixtures as efficient catch crops
We were not able to predict with confidence the amount
of nitrate leached during the growing period under CC
mixtures because the STICS model was not parameterized to simulate the growth and N acquisition of the
species in our bispecific mixtures. However, during the
growing period, model predictions indicated that legume SC effective produced a catch crop service since
they took up more SMN and decreased nitrate leaching
more than bare soil. This conclusion supports the results
found in the literature that indicate legume SC can
reduce about 40 % nitrate leaching, unlike bare soil
(Meisinger et al. 1991; Tonitto et al. 2006). We found
that some mixtures, especially those with turnip rape,
were as efficient as non-legume SC in decreasing SMN
on the date of CC destruction. As demonstrated in the
literature, both species in bispecific mixtures tend to
have deeper and faster root growth (Li et al. 2006;
Tosti and Thorup-Kristensen 2010) than they do in SC,

Plant Soil (2016) 401:347364

which can lead to more efficient SMN uptake than nonlegume SC (Kristensen and Thorup-Kristensen 2000).
Although all CC treatments decreased SMN on the
destruction date more than bare soil, we found no difference between CC treatments at Bignan and Lyon,
which can be explained by early nitrate leaching at the
beginning of the CC growing period due to heavy
rainfall. STICS predicted that leaching started on 1
October at Bignan and 11 September at Lyon, only six
weeks after sowing at both sites. It can be hypothesized
that plant roots did not develop rapidly enough to avoid
nitrate leaching under any treatment, except with turnip
rape, which was found to be a fast-growing species, as is
typical for Brassicaceae species (Thorup-Kristensen
2001). Nevertheless, at the three sites, mixtures kept
approximately the same amount of NO3 from leaching
after CC destruction as non-legume SC. Bispecific mixtures, even with legume species, can efficiently reduce
nitrate leaching and produce an efficient catch crop
service. This conclusion is consistent with results found
in other studies for mixtures with legumes grown for
grain production or as CC (Hauggaard-Nielsen et al.
2003; Mller et al. 2008; Tosti et al. 2014). However,
in some specific cases, when legume species are very
favored and fix large amounts of atmospheric N, as it
was at Lyon, it can induce large and rapid N mineralization after CC destruction due to a low C:N ratio
(Quemada and Cabrera 1995; Kumar and Goh 2002;
Justes et al. 2009). These conditions could lead, in the
case of heavy rainfall and associated high drainage
during spring, to a large amount of nitrate being leached,
including some nitrate from CC residue N mineralization after destruction. At sites with substantial winter
and spring rainfall (e.g. Bignan, in Brittany) or permeable soil with low water retention (e.g. Lyon, in the
Rhone River valley, with very pebbly soil), the date of
CC destruction must be adapted to the sowing date of
the next crop. In this case, CC destruction should not be
too early in order to keep some N mineralized from CC
residues from leaching in the spring. However, CC
destruction should not be neither too closed to the next
crop sowing in order to avoid pre-emptive competition
for water and N. According to Justes et al. (2012), the
CC destruction should be managed carefully and must
be carried out at least two weeks before the cash crop
sowing. Maintaining the CC until the end of winter which generally corresponds to the end of drainage
period in temperate European climates- may maximize
both green manure and nitrate leaching services

Plant Soil (2016) 401:347364

(Quemada and Cabrera 1995; Kumar and Goh 2002;

Tejada et al. 2008).
Competition and complementarity in CC mixtures
Overall, legume species in mixtures tended to be dominated by non-legumes, except at Lyon. This can be
explained by the influence of environmental factors,
especially soil N availability (Mller et al. 2008), which
was lower at this site due to the very stony soil and a low
water retention; this site also had the lowest SMN at
sowing. Then, the non-legume species appeared to benefit more of facilitation or niche complementarity in
mixtures than legume species which have lower CGR.
This was especially true for turnip rape (Brassicaceae)
which had the highest partial LER, but which negatively
affected the legumes associated indicating low complementarity between the two species. The literature reports
similar results for mixtures composed of a Brassicaceae
species and a legume species (Szumigalski and Van
Acker 2008; Wortman et al. 2012b). Overall, in our
study, CC mixtures used abiotic resources more efficiently than SC to acquire N (i.e. LER >1), as also
observed by Wortman et al. (2012b) and Smith et al.
(2014). This was particularly true at the two sites where
SMN availability was low at sowing. Similar conclusions have been made for cash crops in mixtures, especially intercropped cereal and grain legumes, such as
durum wheat and winter pea (Bedoussac and Justes
2010a). The performances of mixtures confirm the beneficial effect of complementarity between their legume
and non-legume species, especially in resource-use efficiency, mostly due to the principle of niche separation
(e.g. Jensen 1996b).
Production of ecosystem services and interactions
between species
In temperate European climates such as in France, during summer sowing and autumn destruction, some
bispecific mixtures simultaneously produce both ecosystem services, while others mostly favor one of the
two services. For example, at Auzeville, mixtures with
turnip rape reached a high level of catch crop service. At
Lyon, the best catch crop mixtures induced a negative or
neutral green manure service. This could be due to
strong competition by the non-legume species (especially for the foxtail millet/lentil mixture) leading to a high
C:N ratio of the mixture and high N immobilization after

361

incorporation and finally leading to decrease the amount

of N available for the next cash crop in comparison to a
bare soil.
The mixtures that best provided the two services may
have benefited from facilitation processes or niche complementarity for water and nutrients. For example, at
Bignan, the turnip rape associated with faba bean had a
higher N concentration in its shoots than the faba bean,
which could be induced by indirect N transfers from the
faba bean to the turnip rape because of N
rhizodeposition, as demonstrated in the literature for
other species (Stern 1993; Jensen 1996a; Xiao et al.
2004). Our results were consistent with those of
Jamont et al. (2013), who observed higher N accumulation in cruciferous species associated with faba bean
than when the former were in SC, which demonstrated
good niche complementarity for N. This would induce
better N nutrition for the cruciferous species, which
could then lead to a low C:N ratio for the mixture and,
consequently, produce a green manure effect nearly as
high as that calculated for the legume SC.
Finally, the integrative effect of both catch crop and
green manure services highlighted the interest of such
CC legume/non-legume mixtures for having more regularly a positive impact of CC whatever the soil type and
climatic conditions which determine the drainage and
nitrate leaching and then the pre-emptive competition of
CC for N. Globally, CC mixtures preserve N into the
soil-crop system thanks to N acquisition by plants and
then lead concomitantly i) to reduce nitrate leaching and
ii) to increase N release for the next crop thanks to CC
residues mineralization. The variability in the mixtures
behavior can be used for choosing the most adapted
mixture to the situation. Indeed, estimating the degree
to which both services are provided could help in
selecting the most suitable species according to the risks
of leaching and pre-emptive competition, which depend
on fallow period management and soil and climate
conditions. For example, it could be more beneficial to
sow a mixture favoring the green manure effect on a site
with low SMN availability and low autumn and winter
rainfall (e.g. Auzeville site). Conversely, it would be
recommended to sow mixtures favoring the catch crop
effect on a site with high SMN availability, a permeable
soil (pebbly, sandy) and high rainfall (e.g. Bignan and
Lyon). Our results are relevant for a growing period of
approximately 34 months during summer and autumn;
however, interactions within mixtures evolve over time
(Bedoussac and Justes 2010b). It would be interesting to

362

investigate the performances of CC mixtures for a longer period (e.g. including CC destruction after winter
and before spring crop sowing) before generalizing
conclusions to fallow periods of all durations.

Conclusion
Bispecific CC mixtures composed of a legume and a
non-legume can simultaneously provide green manure
and nitrate catch crop services more efficiently than
non-legume SC, and at approximately the same level
as them, by taking up SMN and reducing nitrate
leaching. Performances of CC mixtures were optimized
due to good complementarity between legumes and
non-legumes, which together make better use of resources than SC, especially in systems with low N
availability. The mixtures tested in our experiments
provided compromises between the two ecosystem services of nitrate capture and green N manuring, which
allows the choice of mixture to be adapted according to
a sites soil and climate conditions, the priorities of
fallow-period management, and the services desired.
Acknowledgments This study was supported by the Arvalis
Institut du Vgtal, the Midi-Pyrnes Region, the UMR AGIR of
INRA and the French National Research Agency (project ANR09-STRA-06; MicMac-design, Programme STRA 2009). The
authors would like to thank M. Labarrre and L. Bossut for their
efficient technical help and A. Gavaland, P. Bataillon and D.
Campergue at the INRA Auzeville experimental unit for technical
assistance. We also thank E. Masson, M. Moquet, V. Bouetel, D.
Millet and S. Lair at the Arvalis Bignan research site and Y.
Pousset, J. Pauget and A. Authier at the Arvalis Lyon research
site. We thank M.L. and M.S. Corson for improving the English in
the text.

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