Download Soil type determines how root and rhizosphere traits relate

Plant Soil
DOI 10.1007/s11104-016-3127-3
REGULAR ARTICLE
Soil type determines how root and rhizosphere traits relate
to phosphorus acquisition in field-grown maize genotypes
Ran Erel & Annette Bérard & Line Capowiez & Claude Doussan & Didier Arnal &
Gérard Souche & André Gavaland & Christian Fritz & Eric J. W. Visser & Silvio Salvi &
Chantal Le Marié & Andreas Hund & Philippe Hinsinger
Received: 9 May 2016 / Accepted: 24 November 2016
# Springer International Publishing Switzerland 2016
Abstract
Aims Phosphorus (P) is frequently limiting crop production in agroecosystems. Large progress was achieved in
understanding root traits associated with P acquisition
Responsible Editor: N. Jim Barrow.
Electronic supplementary material The online version of this
article (doi:10.1007/s11104-016-3127-3) contains supplementary
material, which is available to authorized users.
R. Erel : D. Arnal : G. Souche : P. Hinsinger
INRA, UMR Eco&Sols, Place Viala, 34060 Montpellier, France
A. Bérard : L. Capowiez : C. Doussan
INRA, UMR1114 EMMAH, Site Agroparc, 84914 Avignon,
France
A. Gavaland
INRA, UE Auzeville, 31326 Castanet Tolosan, France
C. Fritz : E. J. W. Visser
Department of Experimental Plant Ecology, RUN-Radboud
University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen,
The Netherlands
S. Salvi
Dipartimento di Scienze Agrarie (DipSA), Universita’ di Bologna,
viale Fanin 44, 40127 Bologna, Italy
C. Le Marié : A. Hund
Institute of Plant Science, ETH Zurich, LFW A8,
Universitaetstrasse 2, 8092 Zurich, Switzerland
Present Address:
R. Erel (*)
Gilat Research Center, Mobile Post Negev, 85-280 Negev, Israel
e-mail: [email protected]
efficiency (PAE, i.e. P uptake achieved under low P
conditions). Most former studies were performed in
controlled environments, and avoided the complexity
of soil-root interactions. This may lead to an oversimplification of the root-soil relations. The aim of the
present study was, therefore, to identify the dominant
root and rhizosphere-related traits determining PAE, in
contrasting soil conditions in the field.
Methods Twenty-three maize hybrids were grown at
two contrasting P levels of a long-term P-fertilizer trial
in two adjacent soil types: alkaline and neutral. Bulk
soil, rhizosphere and root parameters were studied in
relation to plant P acquisition.
Results Soil type had robust effect on PAE. Hybrids’
performance in one soil type was not related to that in
the other soil type. In the neutral soil, roots exhibited higher
specific root length, higher root/shoot ratio but lower PAE.
Best performing hybrids in the neutral soil were characterized by top soil exploration, i.e., greater root surface and
topsoil foraging. In contrast, in the alkaline soil, PAE and
foraging traits were not correlated, P availability in the
rhizosphere was greater than the bulk soil and phosphatase
activity was higher, suggesting a ‘mining strategy’ in that
case (i.e. traits that facilitate elevated P availability).
Conclusions These results indicate the key role of environmental factors for roots traits determining high
PAE. The study highlights the need to consider soil
properties when breeding for high PAE, as various soil
types are likely to require different crop ideotypes.
Keywords Phosphorus acquisition . Rhizosphere . Root
morphology . Root architecture . Maize
Plant Soil
Introduction
Phosphorus (P) is an essential macronutrient with a
central role in numerous structural and biochemical
plant processes. Due to strong P fixation to soil constituents, P is limiting crop production in many arable soils
and P fertilizer application is frequently needed to
achieve high productivity (Hinsinger 2001; Lynch
2007). Rock phosphates that are mined for elaborating
P fertilizers are a finite and depleted resource (Penuelas
et al. 2013), and thus their prices are expected to further
rise in the coming decades (Brunelle et al. 2015). Plants
have developed numerous P acquisition means to access
and release soil P. In the course of modern plant breeding some of those adaptive traits have been lost
(Wissuwa et al. 2009). Therefore, there is a need to
develop future crops with superior root traits for a better
acquisition of P from soils (Lynch 2007).
The acknowledged root traits enabling soil P acquisition can be classified into two main strategies: ‘foraging’ and ‘mining’. Foraging strategy is based on root
traits related to enhancement of soil exploration at minimal metabolic cost, such as reduced root diameter.
Mining strategy is based on the collection of
rhizosphere-related root traits elevating the availability
of bound P, mainly by root exudation and a whole range
of P-mobilizing rhizosphere processes (Richardson et al.
2011). An additional key player for plant P acquisition is
the rhizosphere microbiome (Bais et al. 2006;
Richardson and Simpson 2011), and specifically the
symbiotic microorganisms in it, such as arbuscular mycorrhizal fungi (Treseder 2013).
Due to the poorly mobile nature of soil P, increasing
the soil volume explored by roots (foraging strategy)
will increase the interacting absorptive surface area and
thus, the size of the potentially accessible pool of P. To
do so, plants have the capability to alter root morphology and architecture to maximize their root surface and
adjust it to the spatial distribution of soil P (Lynch 2011;
White et al. 2013; Péret et al. 2014). Root traits related to
foraging include a higher relative investment of carbon
to the root, occasionally called root mass fraction, i.e.
roots/total plant biomass. Additional foraging related
traits includes branching and elongation of laterals at
the expense of primary root growth, production of finer
roots, and the development of longer root hairs at greater
density (Lynch 2007; Lynch 2011; Rose et al. 2013; Zhu
and Lynch 2004). As a result, typical adaptation of the
root system to P-deficiency is an increase in root length
per unit of root mass, commonly called specific root
length (SRL, i.e. total root length divided by total root
biomass). However, such morphological adaptations are
not universal across plant species (Lambers et al. 2006).
To reduce the carbon and P costs of root exploration, the
development of aerenchyma is another potentially beneficial trait (Lynch 2007). Modification of root architecture
is another typical response to limited P supply
(Weerarathne et al. 2015). Hence, the root openingangle (i.e. the angle between the outmost roots), is considered to have positive impact on soil P acquisition,
especially in those soils exhibiting a strong vertical gradient of P availability (Lynch 2011; Lynch and Brown
2001). In such soils, especially as a result of high fertilizer
application or accumulation of leaf litter, P concentration
tends to be higher in the topsoil, and thus topsoil foraging
may contribute to a high P acquisition efficiency (PAE,
i.e. P uptake achieved under low P conditions) (Lynch
and Brown 2001; Richardson et al. 2011; Wissuwa et al.
2009). Symbiosis with mycorrhizal fungi also increases
the volume of soil explored by roots through the extension of hyphae (Richardson et al. 2009; Shenoy and
Kalagudi 2005) and therefore may also be accounted
for as an attribute of the foraging strategy.
The second P acquisition strategy (mining) is
targeting the enhancement of P availability in the vicinity of the root. The main processes for mining sparingly
available P are the production and exudation of protons/
hydroxyls, carboxylates, phosphatases and other root
exudates (Bais et al. 2006; Hinsinger 2001; Lambers
et al. 2006). Exudation of carboxylates was reported to
potentially increase the solubilization of P compounds
under P deficiency (Bolan et al. 1994; Veneklaas et al.
2003), depending on the carboxylate and soil type
(Duputel et al. 2013). Carboxylate exudation is
displayed by most plant species, although at considerably varying levels (Neumann and Römheld 1999). All
plant species seem to be capable of altering rhizosphere
pH (Hinsinger et al. 2003), which has a major impact on
soil P availability (e.g., Devau et al. 2011a; b). The
combined effect of organic exudates and pH modification may further improve PAE under P-limited conditions, either directly by dissolving soil-bound P, or indirectly, by altering microbial activity (Bais et al. 2006;
Hinsinger et al. 2009; Hinsinger et al. 2003). A large
portion of soil P occurs in organic forms, and a common
response of plants and microorganisms to P-deficiency
is the synthesis and exudation of acid phosphatase enzymes (Richardson and Simpson 2011; Vance et al.
Plant Soil
2003), while alkaline phosphatase enzymes are being
produced and released only by microorganisms (George
et al. 2002). However, the quantitative contribution of
microbial activity to P availability under field conditions
is still debated (Richardson and Simpson 2011).
Growing cereals accounts for more than half of
world’s P fertilizer consumption, and maize takes a large
part of this (Heffer 2009). For environmental, economic
and social reasons, low input maize production systems
are dominant. In such agroecosystems, a high PAE is
essential for sustainable production in P-depleted soils.
Maize is generally considered as a species susceptible to
P deficiency, compared to soybean and sunflower, partly
as a result of lower SRL, a less shallow root system and
minor development of aerenchyma (Fernandez et al.
2009; Fernandez and Rubio 2015). On the contrary, in
a long-term experiment, Colomb et al. (2007) reported
that maize had the lowest Olsen P threshold value,
compared to wheat, sunflower, sorghum and soybean.
The authors mentioned the soil type and cropping system as major sources of variation in PAE under Plimited conditions of these crop species.
Soils are highly heterogeneous environments that interact with roots and may have numerous implications on
root morphology, architecture and physiological performance (Bengough et al. 2006; Hinsinger et al. 2009).
Chemical and physical properties may vary considerably
within a soil, even at short distances, e.g., at the metric
scale within a field (Fu et al. 2013a; b). In particular,
nutrients tend to be unequally distributed (Rose et al.
2013). Physical properties of soil may have large impact
on root system traits related to P acquisition (Hodge et al.
2009). Due to soil-root interactions, inconsistent responses of root system performance in artificial media
versus the soil environment are frequently found (Haling
et al. 2010; Zhu and Lynch 2004). Lambers et al. (2006)
illustrated the dependence of various Western Australian
plant species differing in P acquisition strategy to soil
conditions. The distribution pattern of the plant species
indicated an association between P acquisition strategy
and soil properties, suggesting that the complex relations
between roots and soil should be studied to uncover the
advantage of certain strategies to specific soil conditions
(Lambers et al. 2006).
Although substantial progress in acquiring new
knowledge on PAE under P-limited conditions in crops
has been made in recent years, a large portion of the
research was carried out in artificial, controlled conditions, which allowed focusing on well-defined variables
that are more difficult to measure on soil-grown plants.
However, there is a need for more holistic approaches to
evaluate the large range of P acquisition traits under
field conditions for various soils in order to validate
the data obtained in laboratory and climate-controlled
room. This more complex integration requests a multidisciplinary effort which incorporates, rather than avoid,
the challenges of working with soil-grown plants
(Wissuwa et al. 2009). Therefore, the aim of the present
study was to identify functional root and rhizosphererelated traits that contribute the most to P acquisition
under field conditions. To accomplish this, a field experiment was conducted in a long-term P fertilizer trial
exhibiting contrasting P availabilities and soil types.
Materials and methods
Experimental site and soil types
The experimental site was located at the INRA Research
Centre of Toulouse-Auzeville, Southern France
(43.53oN, 1.505°E), which has traditionally been a common area for maize cultivation. The alluvial soil has a
silty-clay texture, classified as a Luvisol by Devau et al.
(2011a). This site was used for long-term P studies that
were initiated in 1968. Detailed initial soil properties
and cropping history are given by Colomb et al. (2007).
The original experimental site was composed of four
levels of P application rate in four replications, arranged
in a randomized block design. The four P levels at the
time of the current experiment (and applied since 1994)
were: 0, 11, 22 and 33 kg P ha−1 year−1 for P0, P1, P2
and P4, respectively. The current study focused on the
two extreme P levels: P0 and P4. Phosphorus was
applied manually as superphosphate before sowing.
Each of the 16 subplots (4 P levels × 4 blocks) was
30-m long by 6-m wide. In the present work, we focused
on the two extreme levels (P0 and P4) and more so on
the P0 treatment, which had not been fertilized since
1968, in order to properly evaluate the PAE (P uptake
achieved under low P conditions) of maize hybrids.
In spite of the small dimensions of the experimental
site (approximately 100 × 30 m) and the prolonged
history of well controlled cultivation practices and P
application (Colomb et al. 2007), the variability of P
availability within the high P level (P4) was very large.
Olsen P range varied from ~5 to 38 mg kg−1 (Fig. 1),
probably explained by variation of calcium carbonate in
Plant Soil
P). In the high P level, however, P availability was
markedly and significantly higher in the neutral soil, for
both Olsen P and water P. The concentration of CaCO3,
the resulting HCO3− concentration and pH values were
somewhat higher in the alkaline soils of the P0 treatment,
compared to P4 because the latter actually exhibited a
broader range of values, one of the plots being at the
transition between the two soil types, as shown for
HCO3− concentrations in Fig. 1.
Experimental design and agricultural practices
Fig. 1 Inorganic extractable P (Olsen P) as a function of bicarbonate concentration in soil water extract (1:10) for the two P
levels: P4 (squares) and P0 (circles). The data oriented from soil
classified as Balkaline^ are denoted by filled symbols and from the
Bneutral^ soil in open symbols
the soil (Table 1; Colomb et al. (2007)). In the low P
level (P0), on the other hand, P concentration was low and
was not related to bicarbonate concentration (Fig. 1). Regarding the alkalinity (expressed as HCO3− concentration
in 1:10 extraction), two clearly distinguished groups could
be observed: below and above 1.0 mM HCO3− (Fig. 1).
This value was the base of classification of the experimental site into two groups: alkaline soils (i.e.
HCO3− > 1.0 mM) and neutral soils (i.e. HCO3− < 1.0 mM).
Each of these soil classes were arranged to contain two
subplots of 23 hybrids × 2 P levels. These two classes will
be referred as soil type henceforth.
The average values of some chemical soil properties in
each soil type and P level are presented in Table 1. For the
low P level, extractable P concentration was not modified
by soil type in spite of the large variation in CaCO3
concentration and consequently, in the pH of soil water
extract and bicarbonate concentration (Table 1). This was
observed for both Olsen P and water-extractable P (water
To evaluate a wide variation in root traits, 23 maize
hybrids with potentially contrasting root systems were
used. The hybrids were crosses of the parent UH007 with
the lines B73, UH250, Mo17, EC169, F98902, FV353,
MS153, F7028, EZ47, EZ37, Os420_RootABA1-,
Os420_RootABA1+, EZ11A, LH38, Pa405, Oh33,
OH43, FC1890, F912, W64A, B84, MS71, LAN496
(hybrids 1–23, respectively), produced by DSP Ltd.,
Delley, Switzerland. Each block was composed of two
plots (one for each P level) with eight rows (75-cm interrow) of ~24 m in length excluding the boundaries and
irrigation path, which divided the plots in two. The two
marginal rows were excluded and a commercial maize
hybrid (Jumper) was sown there to reduce boundary
effect. The subplots were composed of single hybrid
(except B73 which was repeated twice) sown in 6 m
continuous row (~24 plants). Soil was tilled according
to local practice: deep ploughing (28–30 cm) during
previous October then shallow tillage (8–10 cm) with
power harrow on 24th April 2013. Seeds were sown on
7th May. Weeds were controlled by glyphosate application (900 g ha−1) on 16th April and post-sowing herbicide
application (1340 g ha−1 S-métolachlore +250 g ha−1
Table 1 Average and range (in brackets) of chemical properties of the two soil types for the two studied P levels. The effect of soil type was
analyzed by one-way ANOVA (n = 48)
P0
P4
Olsen P
(mg kg−1)
Water P
(mg kg−1)
CaCO3
(%)
Soil pH
HCO3−
(mM)
Alkaline
2.80 (3.75)
0.42 (1.06)
4.20 (3.03)
7.70 (0.50)
2.39 (1.25)
Neutral
2.81 (3.87)
0.46 (1.13)
0.12 (0.09)
6.69 (1.12)
0.24 (0.80)
p value
0.9866
0.3586
<0.0001
<0.0001
<0.0001
Alkaline
13.4 (26.3)
1.09 (2.23)
2.23 (5.08)
7.50 (0.66)
1.74 (2.45)
Neutral
23.0 (35.6)
1.48 (2.97)
0.23 (0.23)
6.84 (1.23)
0.31 (0.97)
p value
<0.0001
0.0022
<0.0001
<0.0001
<0.0001
Plant Soil
aclonifen +37.5 g ha−1 isoxaflotole). Nitrogen was applied as ammonium nitrate at a rate of 175 kg N ha−1
(105 kg N ha−1 on 14th June +70 kg N ha−1 on 5th July).
Pests were controlled by Lambda cyhalothrine application (25 g ha−1) on 27th June. To avoid severe drought,
103 mm of supplementary irrigation was applied in three
applications, 31 mm on 18th July (in between the Stage-I
and Stage-II harvests as described below), 38 mm on 26th
July and 34 mm on 5th August 2013.
roots in the topsoil, we quantified the root pixels in the
upper 30° and divided it by the total number of root pixels
(see picture). The use of the REST picture analysis tool
made the process fast and efficient. In the picture analysis
tool ‘root depth’ was defined as the depth at which 90% of
the excavated root system was found. The opening angle
was obtained by REST automatic measurement of the
angle between the outmost roots seven cm from the base.
Plant and soil chemical analysis
Stage-I (V6-V7) harvest
Representative plants were harvested on 17–20
June 2013 at V6-V7 stage (6–7 expanded leaves). For
each subplot, six plants per hybrid were excavated with
a shovel, together with their surrounding soil. Soil was
separated into two categories: bulk soil and rhizosphere.
Bulk soil was collected from loose soil free of roots, and
rhizosphere from the thin soil layer firmly adhering to
the roots. The soils collected from the six plants were
combined. One subsample was kept at 4 °C for microbial activity measurements. Another soil subsample was
air dried, ground to 2 mm, and kept at room temperature
until chemical and biochemical (phosphatase activity)
analysis. Roots and shoots were separated and weighed.
Shoots were dried at 60 °C, weighed, ground and stored
for mineral analysis. The six root systems of hybrid x
subplot were combined. The roots were scanned (Epson
11000xl flatbed scanner, Epson Inc., Long Beach, CA,
USA) and analyzed with root morphology analyzing
software (WinRhizo Pro 2005b, Regent instruments,
Montreal, QC, Canada). Representative root subsamples
were dried to determine root water content. Root dry
mass and root length were later used for calculation of
SRL by dividing the total root length by the root dry
biomass of the whole root system (m g−1).
Stage-II (VT) harvest
Plants were harvested on 22–25 July 2013 at VT
(flowering) stage. For each subplot, three representative
plants per hybrid were excavated according to the
‘shovelomics’ approach as described in Trachsel et al.
(2 011 ), although on smaller soil b locks
(25 cm × 25 cm × 25 cm). Shoots were dried, weighed
and ground for chemical analysis. The cleaned topsoil root
system was split into two and photographed in a custom
tool described in Colombi et al. (2015), and the pictures
were analyzed accordingly. To determine the fraction of
Plant mineral analysis was performed on the combined
six plant shoots in stage-I and on the combined three plant
shoots in stage-II. Approximately 100 mg of plant shoot
powder were digested in a microwave oven (Ethos Touch
Control, Milestone) with concentrated HNO3 at 180 °C at
2 MPa. Phosphorus concentrations in the digests were
measured by the vanado-molybdate method. Blanks and
reference materials of maize leaves (V 463, Bureau
Interprofessionel d’Etudes Analytiques, France) were included during the digestions and analyses to check the
accuracy of the measurement procedure. Phosphorus uptake was calculated by multiplying shoot biomass by
shoot P concentration (thus excluding the roots).
The pH of soil water extracts, HCO 3− , waterextractable P (water P) and Olsen P were measured for
each hybrid x subplot, (i.e., four per hybrid x P level,
n = 196). CaCO3 was measured in two repetitions per
subplot (n = 16). The P concentration (water P) and pH
of soil water extracts was determined in 1:10 soil:water
suspension after shaking for 30 min. HCO3− determination was performed on similar soil solution by titration
with 5 mM H2SO4 using phenolphthalein and methyl
orange as pH indicator (USDA 1954). The CaCO3
concentration was measured by evaluating the volume
of emitted CO2 after dissolution with added HCl according to the NF ISO 10693 standardized method in 2011.
Soil P availability was determined by NaHCO3 extraction using the modified BOlsen^ procedure (Olsen et al.
1954). The P concentrations in the water and Olsen
extracts were measured colorimetrically with the malachite green method. Acid and alkaline phosphatase activities were measured according to Tabatabai and
Bremner (1969). Soil microbial activities were assessed
in fresh soil subsamples with the MicroResp™ technique consisting of a 96-deep-well microplate filled
with soil and with the addition of water only (Basal
Respiration) or seven aqueous carbon substrates (Substrate-Induced Respiration, SIR), sealed individually to
Plant Soil
Plant Soil
R Fig. 2
Box plot of inorganic P availability, soil extracts pH, and
phosphatase activity in the rhizosphere (open boxes) compared to
the adjacent bulk soil (grey boxes) for the alkaline and neutral soils
at the two P levels. In the box, solid lines represent the median
while dashed lines represent the average. Data were also analyzed
by one-way ANOVA, different letters below the boxes indicating
significant differences at p < 0.05
a colorimetric CO2-trap microplate and incubated in the
dark at laboratory temperature for 6 h. Glucose-induced
respiration was used as a proxy of active microbial
biomass (Bérard et al. 2012).
Data statistical analysis
All data were analyzed by JMP 7 statistical package
(SAS Institute Inc., Cary, NC, USA) and regressions
were tested by SigmaPlot 12.5 software (Systat Software Inc., San Jose, CA, USA). In general, the experiment is composed of the three key factors: (1) Soil type
(nominal: alkaline or neutral), (2) P level (nominal, high
or low), and (3) hybrid lines (nominal, 23 hybrids).
Linear correlations between Olsen P and HCO3− and
between P uptake and root properties at the first and
second stage were analyzed separately for each soil type
and P level, given that our primary interest was to
evaluate the determinant of PAE in environments
exhibiting low P availability , i.e. plant performance
under low P conditions (P0). When significant, the p
Table 2 Microbial activity in the rhizosphere versus bulk soil as
affected by soil type at the two P levels. Data was analyzed by twoways ANOVA. The comparison of mean values within P level was
P0
Alkaline
Neutral
P4
Alkaline
Neutral
Effects test:
value associated to this relationship is appearing in the
chart. Non-significant correlations are marked in dashed
regression lines. The effect of soil type on soil properties
was tested using one-way ANOVA for each P level
separately. The effect of soil type on SRL was tested
using one-way ANOVA. The effect of rhizosphere versus bulk soil on soil P availability (Olsen P), phosphatase activities and microbial activities was tested using
one-way ANOVA for each P level separately.
To identify the factors governing plant growth, P
uptake and morphological roots traits, analysis of covariance statistical model (ANCOVA) was assigned. In
the ANCOVA, the different measured plant traits were
modelled as depending on soil type (2 factor levels),
hybrid (23 factor levels) and plot-specific Olsen P as
continuous covariate. Olsen P was selected due to random variation in Olsen-P within given P level and soil
type. When taking into account the effect of hybrids, the
random variation in Olsen-P was found to significantly
correlate to P uptake and biomass production. Hence,
Olsen-P was chosen as soil characteristics covariant in
the model to account for the soil heterogeneity. For
simplicity and because our major focus was to assess
PAE under P-limited conditions, i.e. crop performance
at low P, the ANCOVA statistical model was performed
for each P level separately.
To study the effect of soil type on root architecture, a
two-way ANOVA model was assigned evaluating soil
type, hybrid and interaction as explanatory factors for
made by Tukey’s honest significance test, different letters indicating significant difference at p < 0.05
μg-C g soil−1
---------------------μg-C g soil−1 h−1--------------------
Microbial biomass
Basal respiration
SIR - glycine
SIR - malate
Bulk
132 b
0.27 b
2.04 b
2.57 d
Rhizosphere
198 a
0.39 a
2.75 a
3.20 c
Bulk
166 ab
0.32 ab
1.32 c
3.84 b
Rhizosphere
199 a
0.33 ab
1.64 bc
4.87 a
Bulk
140 d
0.25 b
2.04 b
2.96 d
Rhizosphere
351 a
0.77 a
4.11 a
5.11 b
Bulk
199 c
0.28 b
1.92 b
4.23 c
Rhizosphere
291 b
0.23 b
2.26 b
6.03 a
P level
***
n.s.
***
***
Soil type
**
n.s.
***
***
P level x soil type
***
***
***
**
Plant Soil
Table 3 Statistical model (ANCOVA, analysis of covariance) of
the P0 plots examining the effects of the soil type and hybrid line,
with Olsen P in the bulk soil as covariate, on plant biomass, P
uptake and some root morphological traits on the V6-V7 stage (I)
and the flowering stage (II). The least square means are normalized
estimates of the means
g (dw)
(mg plant−1)
(g dw)
(cm)
Shoot biomass
P uptake
Root biomass
Root morphology stage-I
Stage I
Stage II
Stage I
Stage II
Stage I
Stage II
Length
surface
Diameter
Alkaline
0.90
34.7
2.75
64.7
0.52
5.40
972
204
0.68
Neutral
0.66
22.7
1.88
37.4
0.52
3.06
1282
224
0.56
Whole model
R2
0.66
0.54
0.54
0.48
0.57
0.57
0.69
0.52
0.64
p
<0.0001
<0.0001
<0.0001
0.0011
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
Explanatory factors
Soil type
***
***
***
***
n.s.
***
***
*
***
Hybrid line
***
n.s.
*
n.s.
***
n.s.
***
**
**
Bulk Pi
**
n.s.
**
*
n.s.
n.s.
n.s.
n.s.
*
P0
LS-Means
(cm−2)
(mm)
n.s. not significant, * significant at 0.05 > p > 0.001, ** significant at 0.001 > p > 0.0001 and *** significant at p < 0.0001
each P level separately. The fraction of shallow roots
was calculated from the picture analysis tool (REST) by
dividing the number of root oriented pixels in the upper
30° by all roots oriented pixels (see illustration in
Fig. 4). The effect of P level on biomass, P uptake, roots
traits and microbial activity was tested for each soil type
separately by one-way ANOVA. Results are presented
in the supplementary material (Tables S1 and S2). The
comparison of mean values was conducted by Tukey’s
honest significance test.
soil, Olsen P decreased. Significant rhizosphere acidification was found in the neutral soil at the low P level and
in the alkaline soil at the high P level (Fig. 2), but the pH
of soil water extracts appeared to be comparable at the
remaining comparisons.
Enhancement of enzymatic activity in the rhizosphere (compared to bulk soil) was evident for acid
phosphatase but not for alkaline phosphatase (Fig. 2).
Both phosphatases’ activities, acid and alkaline, were
significantly higher in the rhizosphere of alkaline soil
than in the rhizosphere of the corresponding neutral soil.
Results
Microbial activities in the bulk soil and rhizosphere
Changes of soil P availability, pH and phosphatase
activities in the rhizosphere
Overall, microbial biomass and catabolic activities were
higher in the rhizosphere compared to the bulk soil at
given soil (Table 2). The high P level was positively
associated with microbial activities in the rhizosphere
but not in the bulk soil (Table S1). For example, per soil
type, microbial biomass was comparable in the bulk soil
of the high and low P levels. In the rhizosphere of the
high P level, microbial biomass was notably higher. The
effect of soil type (alkaline versus neutral) varied, depending on the specific microbial catabolic activities.
Substrate-induced respiration (SIR) using various organic compounds may indicate changes in microbial
(catabolic) activities. For example, when applying an
N-rich substrate such as an amino acid (SIR-glycine),
respiration was higher in the alkaline soil, while for a Crich substrate such as a carboxylate (SIR-malate), respiration tended to be higher in the neutral soil type.
For the low P level, P concentrations in the bulk soil were
identical for the alkaline and the neutral soil type
(2.80 mg kg−1). In spite of the identical P concentrations
found in the bulk soil of P0, Olsen P was significantly
higher in the rhizosphere compared to the bulk soil of the
alkaline soil (3.20 mg kg−1) but not in the neutral soil
(2.63 mg kg−1) (Fig. 2). At the high P level, Olsen P was
comparable in the rhizosphere and bulk of the alkaline
soil and significantly decreasing in the rhizosphere of the
neutral soil, compared to the concentration found in the
bulk soil (Fig. 2). Results of both P levels indicate considerable soil effect on root-induced modification of the
rhizosphere. In the alkaline soil, Olsen P increased in the
rhizosphere of P0, while in the rhizosphere of P4 neutral
Plant Soil
Table 4 Statistical model (ANCOVA, analysis of covariance) of
the P4 plots examining the effects of the soil type and hybrid line,
with Olsen P in the bulk soil as covariate, on plant biomass, P
uptake and some root morphological traits on the V6-V7 stage (I)
and the flowering stage (II). The least square means are normalized
estimates of the means
g (dw)
(mg plant−1)
(g dw)
(cm)
Shoot biomass
P uptake
Root biomass
Root morphology stage-I
Stage I
Stage II
Stage I
Stage II
Stage I
Stage II
Length
surface
Diameter
Alkaline
1.48
43.8
6.56
122.4
0.49
4.70
855
218
0.79
Neutral
1.33
38.8
5.28
97.2
0.56
5.05
955
220
0.75
Whole model
R2
0.62
0.49
0.50
0.38
0.55
0.42
0.56
0.57
0.45
p
<0.0001
0.0010
<0.0001
0.0547
<0.0001
0.0145
<0.0001
<0.0001
0.0035
Explanatory factors
Soil type
**
*
**
**
n.s.
n.s.
n.s.
n.s.
*
Hybrid line
***
**
**
n.s.
**
*
***
***
*
Bulk Pi
**
n.s.
***
**
n.s.
n.s.
n.s.
n.s.
n.s.
P4
LS-Means
(cm−2)
(mm)
n.s. not significant, * significant at 0.05 > p > 0.001, ** significant at 0.001 > p > 0.0001 and *** significant at p < 0.0001
Finally, basal respiration was enhanced in the rhizosphere in alkaline soils but not in neutral soils (Table 2).
Statistical model
The combined effect of soil type, hybrid and random
variation in soil P availability (here evaluated with Olsen
P in bulk soils) on plant biomass, P uptake and root
characteristics are all presented in Tables 3 and 4 for P0
and P4, respectively. These models do not allow comparison of the two P levels. For P0, the model was highly
significant and captured most of the variability in the
studied plant parameters (except for P uptake at stage-II).
Shoot biomass and P uptake were consistently and significantly higher in the alkaline soil. The superiority of
the alkaline soil was even more pronounced at the
flowering stage. At stage-II, soil type was major determinant of shoot biomass. Unlike shoot biomass, the root
biomass at stage-I was similar regardless of soil type or
Olsen P value in the bulk soil and was affected only by
hybrid line. At stage-II however, root biomass was significantly higher in the alkaline soil and thus the soil type
had become the dominant factor. Although root biomass
was similar at the first stage, root length and surface were
higher and root diameter was thinner in the neutral soil.
In addition to the major effect of soil type on root length,
the hybrids also had a large, significant effect.
Compared with P0, the model for P4 systematically
exhibited lower coefficients of determination. Unlike in
P0, the soil type had lower impact on the plants, yet
shoot biomass and P uptake were slightly higher in the
alkaline soil, and the major determinants were the hybrids and the P availability. Root biomass was comparable in both soil types and was affected only by the
hybrids. Similarly, root length was not related to the soil
type, nor to the soil P availability, but varied amongst
the hybrids. Root diameter however was slightly and
significantly higher in the alkaline soil.
Root morphology at stage-I
Roots at stage-I were classified into two groups: fine
roots (i.e. laterals, root diameter < 1 mm) and coarse
roots (diameter > 1 mm) indicating the main roots axes.
For P0, fine roots length was significantly higher in the
neutral soil while coarse roots were higher in the alkaline soil. At the high P level, soil type had no effect on
root classification (not shown). The SRL was measured
at stage-I only. For the high P level, SRL was nearly
identical in the two soil types, ~17.5 m g−1. Comparable
values were found also in the alkaline soil at the low P
level (18.8 m g−1). In the neutral soil at P0, the SRL was
considerably and significantly higher than the SRL in
the alkaline soil: 24.8 m g−1 (Fig. 3). Interestingly, at that
stage the root biomass was similar for the two soil types
and P levels, 0.512 ± 0.128; 0.518 ± 0.112;
0.560 ± 0.124 and 0.490 ± 0.132 g per plant for P0
alkaline, P0 neutral, P4 alkaline and P4 neutral respectively (Tables 3 and 4). Hence, the modification in root
morphology of plants grown in neutral soil and low P
was characterized by longer and finer roots, with considerably increased total root length and surface, and
Plant Soil
30
Root architecture (according to shovelomics) at stage-II
Relations between root properties and P uptake
At stage-I, the root system of the maize plant is quite
small and easy to excavate, and, thus, we can assume the
sampled root system is a good proxy of the whole root
system. However, as a consequence of the extensive
root system at stage-II, the sampled root system collected by the shovelomics approach is merely a fraction of
the total root system. Thus, results from excavation will
include the topsoil roots only, and, therefore, root area
To evaluate the key root traits determining PAE under Plimited conditions, the relations between root traits at the
two stages and P uptake are presented in Fig. 4. For the
two root classes, at any given value of root length the
shoot P uptake was higher in the alkaline soil than in the
neutral soil, signifying consistently higher PAE under Plimited conditions in the alkaline soil (Fig. 4). For the
alkaline soil, only the coarse (main, diameter > 1 mm)
Table 5: Architecture root properties of maize at flowering stage
as affected by P level and soil type. Data were obtained using
Bshovelomics^ excavation technique combined with REST picture
analysis tool. ‘Shallow roots’ refers to the roots fraction at the
upper 30°. Data were analyzed by two-way ANOVA examining
the effect of soil type, hybrid line and their interaction
Specific root length (m g-1)
decreased average root diameter (Table 3). Root/shoot
ratio was 0.58 in the alkaline soil at low P, as compared
to 0.33 at high P, while it amounted to 0.78 in the neutral
soil at low P, compare to 0.42 at high P (Tables 3 and 4).
and depth are not a precise measurement, but a proxy of
the whole rooting system used for evaluating differences
between treatments.
At the low P level, the root area, opening angle and
depth in the alkaline soil were significantly greater than
in the neutral soil. For root area and opening angle, soil
type and hybrid line had a highly significant effect,
while root depth was significantly related to soil type
only (Table 5). In accordance with P0, at the high P
level, the root area and depth were significantly greater
for the alkaline soil than for the neutral soil. The opening
angle, however, was not affected by soil type but only
by hybrids. The greatest average root depth was found
in the alkaline soil at the P0 level.
At flowering stage, root angle was positively correlated to root biomass at both P levels, P0 and P4
(r = 0.36, p = 0.0003 n = 144), although at P4, soil P
availability was clearly not limiting maize growth. Unlike root opening angle, the relative distribution of roots
in the topsoil was not correlated to root size in P4
(r = 0.24, not significant).
Alkaline
A
25
Neutral
B
20
B
B
15
10
5
0
P0
P4
Fig. 3 Specific root length as affected by soil type and P level of
maize plants grown at two levels of P. Each bar represents an
average of 48 roots sampled in the V6-V7 stage, scanned, and
quantified by WinRhizo. Data were analyzed by one-way
ANOVA, different letters indicating significant differences at
p < 0.05
(cm2)
Root
area
P0
Means
Alkaline 53.4
Whole model
R2
Neutral
Explanatory
factors
Hybrid x soil type
34.8
0.66
(0)
Opening
angle
(%)
Shallow
roots
(cm)
Root
depth
59.2
4.4
15.1
P4 (cm2)
Root
area
(0)
Opening
angle
(%)
Shallow
roots
(cm)
Root
depth
57.5
62.6
3.2
14.8
13.4
50.3
2.6
11.8
49.4
60.9
3.3
0.33
0.63
0.40
0.29
0.33
0.57
0.35
p
<0.0001 <0.0001
0.0294
<0.0001
0.0003
<0.0001
ns
<0.0001
Soil
type
Hybrid
***
***
***
***
ns
ns
***
***
***
***
ns
ns
**
***
*
***
***
ns
ns
ns
ns
ns
ns
ns
n.s. not significant, * significant at 0.05 > p > 0.001, ** significant at 0.001 > p > 0.0001 and *** significant at p < 0.0001
Plant Soil
Fig. 4 Correlations between root morphological traits (V6-V7
stage, Figs. a, b and c) and root architectural traits (flowering
stage, Figs. D, E and F), and P uptake of maize hybrids grown in
alkaline (open symbols) or neutral (filled symbols) low P soil (P0
level). Correlation coefficients and significances appear in the
figures. Non-significant regressions are marked in dashed lines.
The picture shows examples of two contrasting root systems with
respect to shallowness, as extracted from the picture analysis tool
(REST). The two root systems have a roughly similar size (~17 g
fresh biomass)
root length was significantly and positively correlated
with P uptake. In the neutral soil, the two root classes
and the total root length were positively and significantly correlated to P uptake. Yet, the fine (< 1 mm) root
length had weaker correlation to P uptake compared to
the coarse root length.
At the second stage, root area and P uptake were
strongly, positively and continuously correlated in both
soil types. To characterize root architecture, we chose to
use the fraction of roots comprised in the top 30° angle
of the root system (Fig. 4, picture), from horizontal
rather than the opening angle of the root system, since
the opening angle tended to become greater when the
root system biomass was bigger (not shown). This fraction of shallow roots was generally greater in the alkaline soil, (Table 5 and Fig. 4c) but was not correlated
Plant Soil
Fig. 5 Comparison of P uptake
under low P (P0) conditions (i.e.
PAE) of individual maize hybrid
lines between the two soil types at
stage-I (a) and at stage-II b.
Arrow represents the overall
average PAE of the 23 hybrids
with P uptake. On the contrary, in the neutral soil the
fraction of shallow roots and P uptake were significantly
and positively correlated. Roots were also generally
deeper in the alkaline soil and the corresponding trait
was not significantly correlated to P uptake.
Hybrids’ response to soil type
At both growth stages, P uptake was higher in the
alkaline soil under low P conditions (Fig. 5, Table 3
and Table S2), which means that PAE under P-limited
conditions was greater in the alkaline than neutral soil.
On average across all hybrids the effect was significant,
P uptake at the P0 level in the alkaline soil amounting to
2.75 mg plant−1 compared to 1.88 mg plant−1 in the
neutral soil at stage I, while it amounted to 64.7 mg
plant−1 in alkaline soil compared to 37.4 mg plant−1 in
the neutral soil for stage II (Table 3 and Table S2).
Hybrids’ performance under low P conditions (i.e.
PAE) ranking at the two stages varied considerably
(Fig. 5). For example: hybrid 14 was the second best
hybrid for P uptake in the alkaline soil at stage-I, but it
was below average at stage II. On the contrary, hybrid
19 had an average P uptake at stage-I, but the highest
average P uptake at stage-II.
Hybrids also varied greatly in their response to soil
type, e.g., some performed much better in the alkaline
soil and few were not affected. For example, at stage-II,
hybrids 1 and 13 had greater P uptake in the neutral
compared to the alkaline soil, while hybrids 5, 6, 15 and
Plant Soil
19 took up more than the double amount of P in the
alkaline compared to the neutral soil. No particular
relation between hybrids’ performance in the two soil
types was noted. In other words, hybrid performance in
one type of soil did not necessarily predict its performance in the other soil type. The best four hybrids in the
alkaline soil achieving the highest PAE under P-limited
conditions (hybrids 19, 5, 6 and 15, respectively, in
yellow) had low performance in the neutral soil, all of
them falling below the average values of PAE (89.8
compared to 31.1 mg plant−1 in the alkaline and neutral
soil, respectively). The best four hybrids achieving the
highest PAE in the neutral soil (4, 1, 13 and 18, respectively) took up similar amounts of P in both soil types
(51.6 versus 58.4 mg plant−1 in the neutral and alkaline
soil, respectively).
Discussion
Understanding the mechanisms governing P acquisition, and thus PAE (here defined as P uptake under
low P conditions), is still a major challenge in plant
nutrition research in spite of the considerable progress
achieved in the past decade on genetic, molecular and
physiological aspects (for selected recent reviews:
(Lambers et al. 2006; Lynch 2011; Niu et al. 2012;
Péret et al. 2014; Richardson et al. 2011; Simpson
et al. 2011). This progress should promote novel, efficient strategies for increasing plant performance in P
depleted soils. To achieve this, beyond root architectural
and morphological traits, root-soil interactions involved
in P acquisition should be thoroughly investigated
(Wissuwa et al. 2009). The present study demonstrates
that root and rhizosphere-related traits for efficient P
uptake in nutrient limiting environments vary substantially with small variations in soil properties. The present field-study made use of a unique field-phenotyping
platform in which plant genotypes were grown in proximity and identical environment (in terms of weather,
cropping history and fertilizer management) but contrasting soil types: alkaline vs. neutral. The different soil
properties are likely to be stemming from patchy Ca
carbonate distribution which had pronounced effect on
pH of soil water extracts and may have further chemical
and physical implications (Colomb et al. 2007), notably
regarding the forms of soil P (including the forms that
cause P to be least available to plants).
Growth dynamics as affected by P availability and soil
type
Generally, P deficiency was shown to restrict plant
growth in the early developmental stages, while its
negative effect was diminished at more mature stages,
as previously shown for maize (Colomb et al. 2000). In
the low P level, leaves at the young stages exhibited
clear P deficiency symptoms, which almost disappeared
with maturation. An example for the recovering effect is
illustrated by the growth at the two stages: at P4-alkaline
soil, shoot biomass at stage-I was 39% higher than at P0
soil, and just 21% at stage-II. For the neutral soil the
magnitude of recovery between the two stages was less
(50% higher and 41% higher for stage-I and II, respectively), which was probably a result of the restriction in
root development at maturation. The impact of P deficiency on root growth appears to be related to the
severity of the stress. Moderate stress was reported to
stimulate root growth, while more severe deficiency
inevitably impairs root growth (Wissuwa and Ae 2001;
Liu et al. 2004; Rose et al. 2013). The described dynamics are in agreement with our findings in the neutral
soil, as root growth was not impaired at the early stage
but severely decreased at the flowering stage, probably
as a result of the reduced leaf area and assimilation by
then (Mollier and Pellerin 1999; Colomb et al. 2000).
Phosphorus seed reserves have a crucial role in the
initiation of root development (Enns et al. 2006) and
may support plant growth for two weeks, whereas P
reserves are depleted thereafter (Nadeem et al. 2014). In
our experimental conditions, at stage V6-V7, P reserves
and uptake were sufficient to maintain comparable root
growth in the two soil types despite the low P availability (Table 3). This, however, occurred at the expense of
shoot growth, which was substantially reduced at low P,
more so in the neutral soil (Table 3). Shoot growth
inhibition resulted in a considerable increase of the
root/shoot ratio as a response to P deficiency at
this early stage of growth. As P deficiency advanced, root growth in the neutral soil was severely impaired, as evidenced at the flowering stage
from topsoil root system biomass (Tables 3 and 4).
Consequently, the retarded root system explored
less soil and P supply further restricted crop
growth. Hence, the gap between the two soil types
increased with time. Such cascade of events had
been thoroughly described by Wissuwa (2003),
who demonstrated by using a model how minor
Plant Soil
increase in P acquisition may have major consequences on total plant performance under low P
environment. According to Wissuwa (2003), under
low environmental P, uptake is restricted primarily
by the size of the root system, hence, small increase in P uptake will stimulate root growth,
which, in turn, will significantly elevate P uptake.
The dynamics of root growth under P0 conditions
in the present experiment support the described
chain of events. At early stage, root biomass in
the two soil types was similar but P acquisition
was slightly higher in the alkaline soil, which
further stimulated root and shoot growth at the
reproductive stage, while at that stage in the neutral soil root growth was still restricted by the low
P availability. Interestingly, the biggest topsoil root
system at the flowering stage was found at P0 for
the alkaline soil, which exhibited a slightly higher
root biomass than at P4 (Tables 3 and 4).
Soil heterogeneity as a key factor determining crop
performance at low soil P availability
Despite the long-term and thoroughly controlled P application, vast variation was captured in the field, especially at the high P level (Table 1). Nevertheless, at the
low P level, which did not receive any P fertilizer for
45 years, the little variation in soil P availability significantly affected maize growth and P acquisition. According to the statistical model, this spatial variability
of P in P0 had major effect on plant growth, although
typically less than the soil type. Availability of P within
the P0 level (resulting from soil heterogeneity) was
indeed positively and significantly correlated with shoot
biomass and P uptake (Table 3). Such a pronounced
effect of soil P availability demonstrates the importance
of capturing short-distance soil heterogeneity when
conducting a field trial for the purpose of phenotyping
for PAE under P-limited conditions. Thorough soil analysis for each single subplot enabled consideration of
such spatial variability in the statistical model. Detailed
soil analyses are expensive and time consuming, however. Rapid, indirect methods for soil characterization
are therefore needed. In this respect, the utilization of
NIR-spectroscopy seems a promising solution (BellonMaurel et al. 2010). Yet, there are still challenges to
meet, especially when applied to mapping soil P availability (Bogrekci and Lee 2005).
Root morphological traits and P acquisition
Soil physical properties, such as compaction, water
content or oxygen may substantially alter root morphological traits (Bengough et al. 2006). Generally, poor,
dry, compacted soils impair total root length, increase
root diameter and alter root hair length and density. The
effect of chemical soil properties on root development
are not thoroughly established, except for the case of Al
toxicity as related to low pH (e.g. (Haling et al. 2010). In
a study examining eleven P-poor soils, Veneklaas et al.
(2003) found little soil effect on root morphology in
spite of the large impact of soil type on P uptake. In
the present study, root length, surface and average diameter were all significantly influenced by the soil type
at the low P level. Modifications of root architecture and
morphology due to P limitation was reported for several
plants species including the main cereals: rice
(Weerarathne et al. 2015) and maize (Mollier and
Pellerin 1999; Zhu et al. 2005) The neutral soil exhibited
classic root response to P deficiency, which included
increases in total root length, surface, SRL and a decrease in root diameter. These root modifications are
considered to be beneficial adaptations favoring P acquisition due to the increase in the absorptive surface
area of the roots at low metabolic cost (Schachtman
et al. 1998; Lynch 2011). In spite of the greater foraging
capacity of the roots developed in neutral soil at low P, P
uptake was significantly lower than that achieved in the
alkaline soil. The higher root diameter found in the
alkaline soil may imply a contribution of more developed aerenchyma to the higher PAE under P-limited
conditions in the alkaline soil as suggested previously
(Lynch 2007; Fernandez and Rubio 2015). Collectively,
our results indicated pronounced morphological alterations caused by both the soil P availability and soil
type. The maize genotypes grown in the neutral soil had
typical foraging response to low P, which was hardly
evident in the alkaline soil type.
Root architectural traits and P acquisition
The ability of root architecture to respond to the spatial
distribution of nutrients in the soil is a valuable trait (Niu
et al. 2012). This is specifically important for P acquisition, as P is typically unevenly distributed, with tendency to accumulate in the topsoil, especially in fertilized
soils. Shallow rooting architecture (high root angle)
enhances topsoil foraging and is thus considered to have
Plant Soil
a positive contribution to P acquisition in low P soils
(Lynch 1995; Lynch and Brown 2001; Richardson et al.
2011). This was specifically confirmed for maize (Zhu
et al. 2005). Root angle is a common indicator for
topsoil foraging; however, we found that roots with
higher biomass tended to have wider rooting angle and
it was thus not easy to determine which is the cause and
which is the consequence. Hence, we alternatively suggest using the relative abundance of roots in the topsoil
as an indicator of topsoil foraging. Utilization of the
REST picture analysis tool (Colombi et al. 2015) made
this process fast and efficient. The relative distribution
of roots in the topsoil was shown to be positively related
to P uptake at the flowering stage of P-deficient plants,
as reported earlier (Lynch and Brown 2001; Lynch
2007). Yet, we found that this relation was soil dependent, as it was significant in the neutral soil only.
The general response of root properties to P level at
stage-II was dependent on the soil type. In the alkaline
soil, root area was comparable at both P levels, while
root depth and the fraction of shallow roots were higher
at low P. In the neutral soil, however, root development
was impaired at the low P level and the fraction of
shallow roots was lowest. The effect of soil type on root
architecture can be resulting from either the different
chemical or physical soil properties. Yet, it is very likely
that the soil effect on architecture is indirect, resulting
from the variation in P acquisition, which limited the
root development in the neutral soil.
Rhizosphere-related traits and P acquisition
Plant are capable of modifying the soil surrounding the
active root zone (i.e., rhizosphere) in order to favor P
acquisition, mainly by exuding carboxylates or altering
the pH, and microbial and phosphatase activity
(Hinsinger 2001). Those rhizosphere processes determine soil P availability close to the roots. While P
uptake on its own is known to result in a depletion of
soil solution and other easily available P pools, the other
above-mentioned rhizosphere processes can conversely
increase P availability in the vicinity of active roots
(Hinsinger et al. 2011). Devau et al. (2011b) predicted
using a geochemical model an enhanced P availability in
the same neutral soil as the one used in the present work.
In their rhizobox experiment with durum wheat
(Triticum turgidum durum L.), a significant increase of
easily available (water-extractable) P was observed in
the rhizosphere, which could be adequately modelled
only when accounting for the combination of rhizosphere pH increase (as observed in their experiment),
and calcium and P uptake (Devau et al. 2011b). A
substantial increase of both water-extractable and Olsen
P was also reported by Betencourt et al. (2012) in a pot
experiment with durum wheat grown in the same neutral
soil as the one used in the present work, although
significant only at the P0 level. However, in the present
field experiment with maize hybrids, for the neutral soil,
Olsen P slightly decreased in the rhizosphere, this depletion being significant only at the P4 level (Fig. 2).
This pool of available P conversely increased in the
rhizosphere of maize hybrids in the calcareous soil. Contrary to these former works with durum wheat, which
consistently reported a significant rhizosphere alkalization
of the neutral soil (Betencourt et al. 2012; Devau et al.
2011b), no such pH increase was observed in maize
hybrids, but rather a slight and significant pH decrease
at the P0 level (Fig. 2). Collectively, our results do not
show any consistent relationship between rhizosphere pH
and P availability, which points to other root-induced
processes being involved in the control of soil P availability in the rhizosphere of maize in the studied soil types.
Phosphatase enzymes may elevate P availability by
dephosphorylation of soil organic P. Acid phosphatases
have optimum pH level below 7 and may be produced
by microorganisms or plants while alkaline
phosphatases are rather of microbial origin only and
exhibit greater activity at alkaline pH. Alkaline and
acid phosphatases activity can be modified by changes
in soil pH (Dick et al. 2000). In the current study, we
found that acid phosphatase activity was higher in the
rhizosphere, while alkaline phosphatase was comparable in the rhizosphere and bulk soil (Fig. 2), suggesting a
possible direct contribution of roots to the phosphatase
pool, although involvement of acid phosphatases of
microbial origin cannot be excluded (George et al.
2002; Rejsek et al. 2012). While the observed increase
of acid phosphatase activity is consistent with the increase of P availability in the rhizosphere of maize
hybrids grown in the alkaline soil, no such consistency
was found in the neutral soil at P0 (Fig. 2). Interestingly,
at both P levels, highest acid phosphatase and alkaline
phosphatase activities were measured in the rhizosphere
of the alkaline soil, implying a general stimulation of
phosphatases’ activities in the alkaline soil. Such enhancement of enzymatic activity may be partially linked
to the microbial activities in the rhizosphere for alkaline
soils. The differences in basal respiration and catabolic
Plant Soil
profiles (e.g., SIR-Malate versus SIR-glycine) between
alkaline and neutral soils confirm the influence of soil
pH on microbial community functions (C and N cycling) and structure in soils (Wakelin et al. 2008; Ben
Sassi et al. 2012; Creamer et al. 2016).
The comparable microbial biomass and basal respiration in the bulk soil of the two P levels indicates that
microbial activities in the bulk soil were primarily limited
by carbon, as usually expected. In the rhizosphere, labile C
is highly available for microbes (Hinsinger et al. 2009),
alleviating the C limitation observed in the bulk soil. When
comparing microbial activities in the rhizosphere of the
two P levels, considerably higher activities were measured
at the high P level, suggesting that soil microbial communities were probably co-limited: primarily by C and, when
C limitation was removed, by P availability (Griffiths et al.
2012). This may have consequences on the food-webs in
soils (the Bnutrient balance^ approach according to
Pokarzhevskii et al. 2003) and then in return on plant
productivity (Van Der Heijden et al. 2008).
The contrasting results found for rhizosphere changes
in the two soil types imply that the dominant P acquisition
means were different. Possibly, the mining strategy,
which tends to elevate P availability, was more prevalent
in maize genotypes grown in the alkaline soil. The stimulation of acid phosphatase activity in the alkaline soil
may have contributed to the elevated P availability in the
rhizosphere of maize, and may thus have played a major
role in its mining strategy in such soil type.
foraging traits were not correlated. In such conditions,
maize roots exhibited increased P availability (Olsen P)
in the rhizosphere, which is indicative of a ‘mining
strategy’, possibly implying the production of acid
phosphatases as a consistent increase of their activity
was measured in the rhizosphere. Soil properties thus
interact with P acquisition and the strategies by which
plants cope with P limitation, and therefore plant breeding for high PAE should take into account such geneticenvironment interactions. Given the current need for
high throughput field-phenotyping, our work strongly
militates for careful investigation of the short-spatial
scale (metric scale) variability of soil properties in field
plots dedicated to investigate large numbers of genotypes within small experimental plots.
Acknowledgments This research received funding from the
European Community Seventh Framework Programme FP7KBBE-2011-5 under the grant agreement no.289300 (EURoot
project). This work was also partially supported by a Chateaubriand fellowship awarded to Ran Erel from the French Ministère des
Affaires Étrangères (France-Israel scientific exchange program).
We kindly thank the donors of the genetic material: Department of
Agroenvironmental Science and Technologies (DiSTA), University of Bologna, Italy (RootABA lines); Misión Biológica de
Galicia (CSIC), Spain (EP52); Estación Experimental de Aula
Dei (CSIC), Spain (EZ47, EZ11A, EZ37); Centro Investigaciones
Agrarias de Mabegondo (CIAM), Spain (EC169); Misión
Biológica de Galicia (CSIC), Spain, (EP52); University of
Hohenheim, Versuchsstation für Pflanzenzüchtung, Germany
(UH007, UH250); and INRA CNRS UPS AgroParisTech, France
(supply of the remaining INRA and public lines). We also thank
Dr. Hillary Voet for helpful statistical advice.
Conclusions
The current study indicates the intrinsic complexity of
agroecosystems, with numerous uncontrolled factors
affecting plant performance in low P environments.
The surprisingly large effect of rather small changes in
soil properties illustrate the need to further explore P
acquisition mechanisms and evaluate PAE of plants in
natural field environments in order to validate the data
obtained in more controlled laboratory conditions. We
demonstrated that soil properties strongly affected the
dominant root traits determining P acquisition for a
given soil type. Hence, there was not one specific hybrid
or group of hybrids being consistently superior in terms
of P uptake under low P conditions (PAE) in both soil
types. The best performing hybrids in the neutral soil
were characterized by a ‘foraging strategy’, i.e., large
root length and topsoil foraging. In contrast, in the
alkaline soil, the PAE under P-limited conditions and
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