J Agro Crop Sci (2015) ISSN 0931-2250

SALINITY STRESS

Biochar Mitigates Salinity Stress in Potato

S. S. Akhtar1,2, M. N. Andersen2,3 & F. Liu1,2
1 Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, T

astrup, Denmark
2 Sino-Danish Center for Education and Research (SDC), Beijing, China
3 Department of Agroecology, Faculty of Science and Technology, Aarhus University, Tjele, Denmark

Keywords Abstract
adsorption; biochar; growth; physiology;
sodium; yield A pot experiment was conducted in a climate-controlled greenhouse to investi-
gate the growth, physiology and yield of potato in response to salinity stress
Correspondence under biochar amendment. It was hypothesized that addition of biochar may
F. Liu improve plant growth and yield by mitigating the negative effect of salinity
Department of Plant and Environmental
through its high sorption ability. From tuber bulking to harvesting, the plants
Sciences
were exposed to three saline irrigations, that is 0, 25 and 50 mM NaCl solutions,
Faculty of Science
University of Copenhagen respectively, and two levels of biochar (0 % and 5 % W/W) treatments. An
Højbakkeg
ard Alle 13 adsorption study was also conducted to study the Na+ adsorption capability of
DK-2630 T
astrup biochar. Results indicated that biochar was capable to ameliorate salinity stress
Denmark by adsorbing Na+. Increasing salinity level resulted in significant reductions of
Tel.: +45 3533 3392 shoot biomass, root length and volume, tuber yield, photosynthetic rate (An),
Fax: +45 3533 3478
stomatal conductance (gs), midday leaf water potential, but increased abscisic
Email: fl@plen.ku.dk
acid (ABA) concentration in both leaf and xylem sap. At each salinity level, incor-
Accepted March 17, 2015 poration of biochar increased shoot biomass, root length and volume, tuber yield,
An, gs, midday leaf water potential, and decreased ABA concentration in the leaf
doi:10.1111/jac.12132 and xylem sap as compared with the respective non-biochar control. Decreased
Na+, Na+/K+ ratio and increased K+ content in xylem with biochar amendment
also indicated its ameliorative effects on potato plants in response to salinity
stress. The results suggested that incorporation of biochar might be a promising
approach for enhancing crop productivity in salt-affected soils.

specific type of biochar (Liu et al. 2013). Biochar consists

Introduction
Biochar is a charcoal-like material obtained by heating any
organic compound through the process of pyrolysis. It has
received a great interest in agriculture to improve highly
weathered or degraded soil for sustainable crop produc-
tion. Biochar can enhance plant growth by improving soil
physical (e.g. soil water-holding capacity, aggregate stabil-
ity, aeration and bulk density), chemical (e.g. nutrient
holding capacity, EC, pH and CEC) and biological charac-
teristics (e.g. rhizosphere microbial population, microbial
biomass C, N and enzymatic activities) (Lehmann and
Joseph 2009, Sohi et al. 2010). The overall, mean increase
in crop productivity reported in literature with biochar
amendment was about 10 % (Jeffery et al. 2011). However,
some studies have reported negative effect of biochar on
crop productivity, but these were generally restricted to
to large degree of recalcitrant carbon (C) (Cheng et al.
2008), which may remain in the soil for 100–1000 years,
and thus, incorporation of biochar could be an effective
approach to increase soil carbon sequestration, thereby
reducing global greenhouse gas (GHG) emission (Lehmann
2007, Sohi et al. 2010, Chowdhury et al. 2014).
There are many reports describing the role of biochar in
remediation of toxic/contaminated soils (Beesley et al.
2011, Uchimiya et al. 2012a,b, Zhang et al. 2013, Ahmad
et al. 2014, Paz-Ferreiro et al. 2014, Samsuri et al. 2014).
The major mechanism involved in remediation of such
soils with biochar was attributed to its high adsorption
capacity (Samsuri et al. 2014, Zhang et al. 2013). The
adsorption capacity of organic and inorganic contaminants
is determined by the characteristics of biochar (Lehmann
and Joseph 2009), which are influenced by both the type of

368 © 2015 Blackwell Verlag GmbH, 201 (2015) 368–378


feedstocks and the pyrolysis conditions under which the
biochar is manufactured (Chen et al. 2011). The favourable
attributes of biochar in terms of its high adsorption
capacity include its high surface area and cation exchange
properties.
Salinity is one of the major constraints to global
food security. According to a recent estimate, on a glo-
bal scale, more than 1100 million hectare (1128 Mha)
land is affected by salinity (Wicke et al. 2011). Plants
under salinity have to cope with two major stresses,
that is osmotic stress and ionic stress. The osmotic
stress immediately affects plants with rising salt level in
the soil solution surrounding the roots and results in
inhibition of water uptake, cell expansion and lateral
bud development (Munns and Tester 2008). Ionic stress
on the other hand develops gradually when toxic ions
(e.g. Na+) accumulate in excess to levels beyond plant
specific thresholds, leading to increased leaf mortality,
chlorosis, necrosis and decreased activity of cellular
metabolism including photosynthesis (Yeo and Flowers
1986, Glenn et al. 1999, Zahir et al. 2012, Panuccio
et al. 2014). Exclusion of Na+ at the plasmalemma and
inclusion of Na+ in vacuoles represent plant strategies
to realize low cytoplasmic Na+ concentrations (Flowers
and L€auchli 1983). Na+ exclusion is especially impor-
tant when ions are absorbed from external solution
(Schubert and L€auchli 1990). The high salt adsorption
capacity of activated charcoals has long been reported
by Bartell and Miller (1923). Similar to charcoal, bio-
char also possesses high salt sorption potential as
recently noticed by Thomas et al. (2013), and therefore,
it could be used to mitigate the negative effect of salin-
ity either by reducing Na+ uptake or by facilitating
Na+ exclusion or both in plants. However, following
the literature reviewed, very limited information is
available about the effect of biochar incorporation on
salt adsorption and its potential role in mitigating
salinity stress in plants.
Therefore, the objective of this study was to evaluate the
salt adsorption potential of biochar and its effect on
growth, physiology and yield of pot-grown potato under
salinity stress. The results could prove useful for developing
novel management strategies for sustaining crop produc-
tion on salt-affected soils.
Table 1 Physico-chemical properties of biochar
BDwh VS Total-C
Material MC (%, web) (Kg m
3, web) (g kg
1 TS) (g kg
1 TS)
Biochar 19 302 906 782
MC, moisture content; web, wet weight basis; BD, bulk density; VS, volatile solids; NH4+-N, ammonium nitrogen; Total-K, potassium; C/N, carbon to
nitrogen ratio; TS, total solids.
© 2015 Blackwell Verlag GmbH, 201 (2015) 368–378
Biochar Application under Salinity Stress
Materials and Methods
Experimental set-up
The experiment was conducted from March to July 2013 in
a climate-controlled greenhouse at the experimental farm
of the Faculty of Science, University of Copenhagen,
Taastrup, Denmark. One slightly sprouted tuber (Solanum
tuberosum L. cv. Folva) was planted in each plastic pot
(38 cm length, 29 cm width and 19.5 cm height) contain-
ing 20 kg of sandy loam soil. The soil was sieved passing
through a 2-mm mesh and had volumetric soil water con-
tent (vol. %) of 30.0 % and 5.0 % at water-holding capac-
ity and permanent wilting point, respectively.
The biochar (BC) used was a commercial charcoal prod-
uct purchased from Skogens Kol AB, Sweden. It was pro-
duced by heating biomass, a mixture of hardwood (80 %)
and softwood (20 %), at 500 °C through the process of
pyrolysis. It was crushed into fine powder and mixed thor-
oughly in soil before filling the pot at the rate of 5 % by
weight. None biochar-amended pots were served as con-
trols. Fertilizers were uniformly mixed in soil before filling
the pots at rates of 150 kg N, 30 kg P, 220 kg K, 30 kg Mg
and 200 kg S ha
1. The soil was classified as sandy loam,
having pH 7.2, total C 12.5 g kg
1, total N 1.4 g kg
1,
water-soluble P 24 mg kg
1, exchangeable Ca

1
3.0 mmol kg and exchangeable K, Mg and Na
<1.0 mmol kg
1. The basic biochar properties are shown
in Table 1.
Before planting, the seed tubers were kept for 7 days at
12–14 °C with constant dim overhead light (a photosyn-
thetic photon flux density (PPFD) of 100–
150 lmol m
2 s
1) for sprouting. During planting, only
one sprout was retained. The climate conditions in the
greenhouse were set at: 16/14  2 °C day per night air
temperature, 15-h photoperiod and >500 lmol m
2 s
1
PPFD supplied by sunlight plus metal halide lamps.
Salinity treatment
Potatoes are considered as moderately salt sensitive having
salinity threshold of 1.7 dS m
1; therefore, at tuber bulking
stage, the plants were subjected to three mild saline irriga-
tion treatments, that is 0, 25 and 50 mM NaCl solutions
Total-N Exchangeable Total-K EC
(g kg
1 TS) NH4+-N (g kg
1 TS) (g kg
1 TS) C/N pH (dS m
1)
3.4 Not-detectable 13.7 236 7.6 0.71
369
Akhtar et al.
during the treatment period (65 days). There were four
replicates of each treatment. The salinity was developed by
dissolving NaCl in tap water having electrical conductivity
0.6 dS m
1. Total volume of water applied to each pot in
0, 25 and 50 mM NaCl solutions (regardless of biochar
treatment) was 26.8, 21.3 and 19.8 l, respectively, during
the whole treatment period. Irrigation was applied manu-
ally to approximately 90 % of pot water-holding capacity.
Tuber yield, shoot and root growth
Fresh tuber yield was measured immediately after harvest-
ing. Shoot dry biomass was determined after oven drying at
70 °C for 48 h. Roots were carefully separated from the soil
mixture using gentle stream of water. Roots samples were
cut into small pieces, and then, images of subsamples were
scanned with Epson Perfection V700 photo (J221A, Seiko
Epson Corp., Tokyo, Japan) scanner at 400 dpi resolutions
using the WinRhizo root scanning software system (version
2009c; Regent Instruments Inc., Quebec, Canada) at the
automatic threshold selection setting (Bouma et al. 2000).
The images were then analysed for root length and root
volume using WinRhizo software by following instructions
provided by Regent Instruments Inc. (2009).
Physiological measurements
Leaf photosynthetic rate (An) and stomatal conductance
(gs) were determined from upper canopy fully expanded
leaves between 11:00 and 14:00 h with a portable photosyn-
thetic system (CIRAS-2, PP Systems, Hitchin, UK). Mea-
surements were performed on two leaves per plant at a
CO2 concentration of 400 lmol ml
1, 28.5 °C chamber
temperature and 1000 lmol m
2 s
1 photon flux density.
Chlorophyll content index was measured with portable
CCM-200 (Opti-Science, Tyngsboro, MA, USA). Leaf water
potential was determined using a pressure chamber (Soil
Moisture Equipment Corp., Santa Barbara, CA, USA) on
fully expanded upper canopy leaves from 10.00 to 11.00 h.
Stomatal morphology
Stomata imprints were obtained from the abaxial surface of
the leaflets used for gas exchange measurements midway
between the leaflet tip and the base. The leaf surface was
cleaned with a soft brush, and a thin layer of nail polish was
applied on the cleaned area and allowed to dry for 10–
15 min. Subsequently, transparent tape was adhered to the
area and carefully drawn off. Then, the imprint was
attached to microscopic slide with the help of solution tape.
Imprints were photographed through a Leitz DMRD
light microscope (Leica Mikroskopie & Systeme GmbH,
Wetzlar, Germany) with an associated camera (Leica DFC
370
420). A 1-mm2 grid was superimposed on the images for
calculating the stomatal density.
Stomatal density and stomatal size parameters, including
guard cell length (Ls), guard cell pair width (Ws), stomatal
pore aperture length (La) and stomatal pore aperture width
(Wa), were measured (data of Ls, Ws, La and Wa are not
presented) with the images using UTHSCSA ImageTool
software (UTHSCSA ImageTool for Windows version
3.00). Stomatal aperture (SA) (lm2) was then calculated
according to Doheny-Adams et al. (2012) using Eqn 1:
p  Wa  La
SA ¼ ð1Þ
4
where Wa is the pore aperture width and La is the pore
aperture length.
Leaf and xylem sap ABA concentration
To determine leaf ABA concentration, two mature leaflets
(74 days after planting) from each treatment were taken
from upper canopy and wrapped with aluminium foil then
immediately dipped in liquid N and subsequently stored at

80 °C until analysis. Approximately 30-mg leaf sample
was weighed and crushed under liquid N using a mortar
and pestle. The samples were homogenized by shaking for
24 h at 4 °C and then centrifuged at 10 000 g for 5 min at
4 °C. The supernatant was taken into another Eppendorf
vial and stored on ice until analysis, whereas xylem sap was
collected by pressurizing the roots of the uprooted plants
(wrapped in polythene bag) in a Scholander-type pressure
chamber and shoot was detopped at 5–7 cm from stem
base. With the stem stump protruding outside the cham-
ber, pressure was applied slowly until the root water poten-
tial was equalized. The pressure was increased gradually
until it equalled leaf water potential of the plants in order
to obtain a sap flow rate similar to the whole plant transpi-
ration rate to avoid dilution effects (Dodd et al. 2008).
1.0 ml of xylem sap was collected using a pipette from the
cutting surface into an Eppendorf vial wrapped with alu-
minium foil and stored at
80 °C until analysis.
ABA concentration from both leaf extract and xylem sap
was determined by an enzyme-linked immunosorbent assay
(ELISA) using a monoclonal antibody for ABA (AFRC
MAC252) (Asch 2000).
Xylem sap ionic concentration
Xylem sap ionic concentration (anions and cations) was
determined by ion chromatography (Metrohm AG, Heri-
sau, Switzerland). Anions were determined on a Metrosep
A Supp 4 analytical column (4 9 125 mm 1.8 mM
Na2CO3/1.7 mM NaHCO3 eluent), and cations were deter-
mined on a Metrosep C4-100 analytical column
© 2015 Blackwell Verlag GmbH, 201 (2015) 368–378
 Biochar Application under Salinity Stress

(4 9 125 mm, 1.7 mM nitric acid/0.7 mM dipicolinic acid
(DPA) eluent).
Total leaf nitrogen (N) and carbon (C) contents
Total leaf N and C contents (% DW) from finely grounded
dry leaf samples were measured directly with a CHNS/O
elemental analyzer (Flash 2000, Thermo Fisher Scientific,
Cambridge, UK) which operates according to the dynamic
flash combustion technique also known as modified
Dumas method.
Adsorption study
This experiment was carried out in the laboratory at
25  1.5 °C. 0.5 g biochar was transferred into 50-ml
polypropylene tubes containing 40 ml of 0, 25, 50 and
100 mM of NaCl solution with four replicates. The tubes
were then agitated for 24 h on a end-over-end shaker at
45 r.p.m. After shaking, supernatant was removed by cen-
trifugation at 10 000 g for 5 min at 25 °C and clear aliquot
was collected in polypropylene tubes and stored at 4 °C
until analysis. The Na+ and Cl
concentrations were deter-
mined by ion chromatography (Metrohm AG) as reported
for the ionic measurements of xylem sap. The Na+ adsorp-
tion capacity of biochar was calculated by the same equa-
tion (Eqn 2) used by Lo et al. (2012) for determining
adsorption capacity of heavy metal ions by activated
charcoal.
fCi
Cf g
AC ¼ V
m
where AC is the Na+ adsorption capacity of biochar, Ci
(mg l
1) and Cf (mg l
1) are the initial and final concen-
tration of Na+ before and after biochar addition, V is vol-
ume of NaCl solution in polypropylene tubes and m is the
dosage of biochar (g).
Statistical analysis
The experiment was conducted in a fully randomized
design in the greenhouse. The combined effects of salinity
Table 2 Effect of biochar (B) and salinity (S) on shoot, root growth and tuber yield of potato
and biochar were analysed using a two-way ANOVA. Means
were compared at the 5 % level of significance using Tu-
key0 s range test. Linear regression analyses between some of
the variables were made on the data across all treatments.
The results of Na+ adsorption experiment were analysed
using one-way ANOVA. All analyses were performed in R ver-
sion 2.15.0 (R Development Core Team, 2012).
Results
Shoot and root growth and tuber yield
Increased salinity significantly decreased shoot dry biomass
(SDW), root length (RL), root volume (RV), number of
tubers per pot (NT) and tuber yield (TY) (Table 2). Incor-
poration of biochar increased RL and RV significantly
(except RV at 50 mM NaCl) compared to respective non-
biochar controls at each salinity level. Tuber yield was also
positively affected by biochar amendment, but significant
increase was found only at 25 mM NaCl treatment. No sig-
nificant effect was noticed on shoot dry weight and number
of tubers per plant with biochar addition.
Physiological responses
The saline irrigation treatment significantly decreased leaf
photosynthetic rate (An) (Fig. 1a) and stomatal conduc-
tance (gs) (Fig. 1b), whereas intrinsic water use efficiency
(WUEi) (Fig. 1c) tended to be increased (particularly at
50 mM NaCl treatments compared to 0 mM NaCl).
ð2Þ Under salinity, biochar amendment, on the other hand,
had positive effect on all three variables, that is An, gs
and WUEi (except for WUEi at 50 mM NaCl solution) at
each salinity level in relation to respective non-biochar
control treatments. In addition, a significant positive lin-
ear relationship between An and gs was observed across
all treatments (Fig. 2). Midday leaf water potential
(Fig. 3a), xylem ABA concentration ([ABA]xylem)
(Fig. 3b) and leaf ABA concentration ([ABA]leaf) (Fig. 3c)
were all strongly influenced by salinity, but the negative
effect on leaf water potential was partially offset by the
biochar amendment. Similar trend was seen for the ABA

0 mM NaCl 25 mM NaCl 50 mM NaCl P values

Variables B0 B5 B0 B5 B0 B5 S B S9B

SDW (g) 24.8  1.9 23.9  3.7 20.1  0.8 20.9  1.3 17.6  0.7 17.9  1.4 0.011 0.941 0.894
RL (m) 132.9  8.9 176.4  8.2 86.4  10.5 111.1  6.4 54.1  15.6 75.2  12.9 <0.001 0.003 0.548
RV (cm3) 5.6  0.7 7.4  0.8 3.3  0.3 4.6  0.3 3.6  0.6 3.98  0.2 <0.001 0.019 0.502
NT (Pot-1) 17.25  1.6 20.5  4.5 8.0  1.1 10.2  2.3 12.2  1.1 10.5  0.9 0.001 0.511 0.524
TY (g) 863.8  47.9 776.1  39.0 463.6  23.7 663.2  9.5 319.7  32.1 358.3  16.0 <0.001 0.044 <0.001

SDW, shoot dry weight; RL, root length; RV, root volume; NT, number of tubers Pot
1; TY, tuber yield.  indicates S.E.

© 2015 Blackwell Verlag GmbH, 201 (2015) 368–378 371

Akhtar et al.

20 25
(a) B0 B5 S: P < 0.001 B0-S0 B0-S25 B0-S50
16 B: P = 0.004 B5-S0 B5-S25 B5-S50
20
An (µmol m–2 s–1)

SxB: P = 0.222

An (µmol m–2 s–1)

12
15
8
10
4
5 r2 = 0.60***
0
(b) S: P < 0.001
0
200 B: P = 0.005
0 50 100 150 200 250 300
gs (mmol m–2 s–1)

SxB: P = 0.068
gs (mmol m–2 s–1)
150

Fig. 2 Relationship between stomatal conductance (gs) and photosyn-

100
thetic rate (An) of potato leaves under different saline irrigations and
biochar treatments. *** indicates the significance of regression line at
50 P < 0.01.

0
WUEi (mmol CO2 mol–1 H2O)

(c)
S:
B:
0.15
SxB: P = 0.061
0.10
0.05
0.00
0 mM NaCl 25 mM NaCl
Salinity treatment
Fig. 1 Photosynthetic rate (a), stomatal conductance (b) and intrinsic
water use efficiency (WUEi) (c) as affected by saline irrigations and bio-
char treatments. B0 and B5 indicate biochar application at the rate of
0 % and 5 % by weight in soil, respectively. S and B indicate saline irri-
gations and biochar treatments, respectively. Error bars indicate S.E.
(n = 4).
concentrations, where the increase induced by salinity
was moderated by biochar although not significantly in
the case of [ABA]leaf. Significant correlations between gs
and [ABA]xylem, [ABA]leaf and midday leaf water poten-
tial were noticed across all treatments (Fig. 4), while the
coefficient of determination for the linear regressions was
the highest between gs and [ABA]xylem.
Stomatal morphology
The effect of saline irrigation and biochar on stomatal den-
sity and stomatal aperture are shown in Fig. 5a. Both vari-
ables were decreased with increasing salinity levels;
however, biochar amendment positively influenced them at
each salinity level compared to respective non-biochar
control.
P = 0.068
P = 0.030 Xylem sap Na+, K+ and Na+/K+ ratio
Xylem sap Na+ and K+ concentrations and Na+/K+ ratio
were strongly influenced by both saline irrigation and bio-
char amendment (Fig. 6). These xylem ionic concentra-
tions (Na+, K+) were increased significantly with increasing
salinity levels, and their maximum values were found
at 50 mM NaCl treatment. For Na+ concentration and
50 mM NaCl Na+/K+ ratio, interaction of saline irrigation and biochar
was also found significant (P < 0.05). Significant reduction
of both Na+ concentration and Na+/K+ ratio in the xylem
with biochar amendment was noticed only at 25 mM NaCl
as compared to the non-biochar control.
Chlorophyll content index, total leaf N and C contents
Figure 7a depicts the reduction of leaf chlorophyll content
index (CCI) with increasing salinity level. Biochar amend-
ment had significantly negative effect on CCI at each salin-
ity levels compared to respective non-biochar control.
The effects of salinity and biochar on total leaf N and C
concentration are shown in Fig. 7b and c. Saline irrigation
(25 and 50 mM NaCl) reduced both total leaf C and N con-
centrations significantly compared to 0 mM NaCl treat-
ment. Incorporation of biochar significantly reduced leaf N
but had no effect on leaf C content compared to the respec-
tive non-biochar control.
Na+ adsorption
Figure 8 shows significantly higher Na+ adsorption capac-
ity of biochar at different salinity levels (0, 25, 50 and
100 mM NaCl). Maximum Na+ adsorption was found at
the highest salinity level, that is 100 mM NaCl solution.

372 © 2015 Blackwell Verlag GmbH, 201 (2015) 368–378

 Biochar Application under Salinity Stress

1.5 300
B0 B5 (a) B0-S0 B0-S25 B0-S50 (a)
S: P < 0.001
Leaf water potential (-MPa)

1.2 B: P = 0.025 250 B5-S0 B5-S25 B5-S50

SxB: P = 0.515

gs (mmol m–2 s–1)

r 2 = 0.75***
200
0.9

150
0.6
100
0.3
50
0.0
(b) 0
S: P < 0.001
0 500 1000 1500
1200 B: P = 0.001
[ABA]xylem (nM)
[ABA]xylem (nM)

SxB: P = 0.404
900
300
(b)
600
250

gs (mmol m–2 s–1)

300
200

0
(c) 150
S: P < 0.001
[ABA]leaf (pmol g–1 FW)

800 B: P = 0.234
100
SxB: P = 0.494
r 2 = 0.46***
600 50

400 0
0 200 400 600 800 1000
200 [ABA]leaf (pmol g–1 FW)

0 300
(c)
0 mM NaCl 25 mM NaCl 50 mM NaCl
250
Salinity treatment
gs (mmol m–2 s–1)

200
Fig. 3 Leaf water potential (Ѱlw) (a), abscisic acid (ABA) concentration
in xylem sap (b) and leaf (c) of potato plant exposed to different saline
150
irrigations and biochar treatments. B0 and B5 indicate biochar applica-
tion at the rate of 0 % and 5 % by weight in soil, respectively. S and B 100
indicate saline irrigations and biochar treatments, respectively. Error
r 2 = 0.70***
bars indicate S.E. (n = 4). 50

Discussion
Our results indicated that the addition of biochar to sandy
loam soil can mitigate negative effect of salinity on the per-
formance of potato plants. Salt-induced disorder on
growth and physiology of the plants could be alleviated by
biochar amendment due to its high adsorption capacity,
which might have either led to reduced Na+ uptake or
enhanced Na+ exclusion or both from the roots. Based on
the adsorption study (where only pure biochar was used)
and the amount of biochar applied to the soil, it could be
estimated that the added biochar adsorbed 97 % of Na+
from the soil irrigated with 50 mM NaCl solution. How-
ever, one should be aware that the added biochar basically
contributes to the cation exchange capacity of the soil (e.g.
0
0.0 0.5 1.0 1.5
Leaf water potential (– MPa)
Fig. 4 Relationship between stomatal conductance (gs) and xylem ABA
(a), leaf ABA (b) and leaf water potential (c) of potato plants under
different saline irrigations and biochar treatments. *** indicates the
significance of regression line at P < 0.01.
Liang et al. 2006), and therefore, the adsorbed Na+ does
not become unavailable to plants, but the Na+ equilibrium
concentration of the soil solution will be lowered. As Na+ is
not among the cations that are strongly adsorbed to
negatively charged surfaces in the soil (e.g. Scheffer and
Schachtschabel 1979), it would be expected that the
adsorption capacity/durability over time is limited, which

© 2015 Blackwell Verlag GmbH, 201 (2015) 368–378 373

Akhtar et al.

250 250
(a) B0 B5 S: P = 0.003 B0 B5 (a)
S: P < 0.001
Stomatal density (mm–2)

B: P = 0.026 B: P < 0.001

200 200
SxB: P = 0.769 SxB: P = 0.033

[Na+]xylem (mM)
150 150

100 100

50 50

0 0
(b) (b)
S: P < 0.001 S: P < 0.001
Stomatal aperture (µm2)

200 B: P = 0.007 B: P = 0.001

600 SxB: P = 0.576
SxB: P = 0.929

[K+]xylem (mM)
150
400
100

200
50

0 0
0 mM NaCl 25 mM NaCl 50 mM NaCl (c)
S: P < 0.001
Salinity treatment 0.8 B: P < 0.001
SxB: P = 0.042
Fig. 5 Stomatal density (SD) (a) and stomatal pore aperture (SA) (b) in
[Na+]/[K+]

the abaxial leaf surface of potato exposed to different saline irrigations
and biochar treatments. The data were determined based on 12 leaf
impression images (each from one individual leaf and three leaves per
plant of four plants per treatment), and of which, six stomata for each
image were measured. SA was calculated using Eqn 1; SD was calcu-
lated by counting stomatal number of the 12 images and each image
with an area of 0.00625 mm
2. B0 and B5 indicate biochar application
at the rate of 0 % and 5 % by weight in soil, respectively. S and B indi-
cate saline irrigations and biochar treatments, respectively. Error bars
indicate S.E.
is exemplified by the strong increase in [Na+]xylem (Fig. 6)
as well as by the reduced beneficial effect on tuber yield
(Table 2) when increasing the salinity level from 25 to
50 mM. Nevertheless, the mitigation effect, even if transient,
might be of importance in many agricultural areas, for
example in monsoon climates where saline irrigation water
builds up salinity during a dry season and salt is subse-
quently leached by rain during the wet season. Thus, by
adding biochar to avoid or postpone, salinity levels reaching
hazardous levels could have significant implication in prac-
tice (Oster 1994). Besides, addition of biochar can enhance
water-holding capacity of the soil, particularly under
reduced irrigation regimes (Akhtar et al. 2014), which
should also help to mitigate the salt-induced osmotic stress
and ion toxicity to the plants due to the dilution effect.
Often, significantly positive growth response of plant
with biochar addition is expected in degraded, nutrient
poor, highly acidic and low water-holding capacity soils
(Atkinson et al. 2010, Jien and Wang 2013). The emerg-
ing literature also supports this hypothesis, that is the
0.6
0.4
0.2
0
0 mM NaCl 25 mM NaCl 50 mM NaCl
Salinity treatment
Fig. 6 Concentrations of Na+ (a), K+ (b) and Na+/K+ (c) in xylem sap as
affected by different saline irrigations and biochar treatments. B0 and
B5 indicate biochar application at the rate of 0 % and 5 % by weight
in soil, respectively. S and B indicate saline irrigations and biochar treat-
ments, respectively. Error bars indicate S.E. (n = 4).
largest growth response with biochar was observed in
problematic soils (Jeffery et al. 2011). Here, mitigation
of salt stress effects on potato plants grown in sandy
loam soil with normal fertility provides another example
of this trend. These findings are in line with that of Las-
hari et al. (2013), who reported enhanced yield with
biochar under salinity stress.
Plant physiological disruptions due to salinity stress have
been extensively studied (Munns 2002, Munns and Tester
2008, Razzaghi et al. 2011, Ionov et al. 2013, Amjad et al.
2014, Hirich et al. 2014). In the present study, it was found
that salinity caused a significant reduction in stomatal den-
sity in potato leaves. This result is consistent with that of
Shabala et al. (2012), who also reported decreased stomatal
density due to increase in leaf succulence and size of

374 © 2015 Blackwell Verlag GmbH, 201 (2015) 368–378

 Biochar Application under Salinity Stress

50 pavement cells under salinity stress. Moreover, stomatal

(a) B0 B5 S: P = 0.005 aperture was also decreased under salinity stress. In addi-
Chlorophyll content index

40 B: P < 0.001
SxB: P = 0.743
tion, our previous work on tomato plants (Akhtar et al.
2014) also found that both stomatal aperture and density
30 were decreased under reduced irrigation regimes, indicat-
ing both drought and salinity stress tend to decrease these
20
variables. Most interestingly, here we found that incorpora-
tion of biochar increased both stomatal aperture and den-
10
sity implying reduced stress was experienced by the plants.
Plant physiological responses to biochar addition under
0
(b) S: P < 0.001 salinity stress have been recently studied by Thomas et al.
2.5 B: P < 0.001 (2013), but the underlying mechanisms remain elusive.
SxB: P = 0.548 Plants may accumulate some phytohormones (i.e. ABA)
2.0
Leaf N (%)

after immediate exposure of branching roots to higher

1.5 salinity levels (Duan et al. 2013). In accordance with this,
our results showed that saline irrigation resulted in
1.0 increased ABA concentrations in both leaf and xylem sap.
0.5
ABA regulates stomatal morphology (Franks and Farquhar
2001, Sun et al. 2014) which directly affects gs. Moreover,
0.0 we noticed that there was a significant negative relationship
(c) S: P < 0.001
B: P = 0.231
between gs and ABA of both xylem and leaf, confirming
44
SxB: P = 0.613 our earlier findings on tomato under reduced irrigation
where we found similar relation between these variables
Leaf C (%)

38
(Sun et al. 2013), indicating that ABA-based chemical sig-
nalling might have played an important role in inducing
32
stomatal closure in salt-stressed potatoes, although an
26
effect of hydraulic signalling in stomatal regulation could
not be excluded as the relationship between gs and midday
20
leaf water potential was significant as well. Nonetheless, the
0 mM NaCl 25 mM NaCl 50 mM NaCl highest coefficient of determination for the correlation
Salinity treatment between gs and [ABA]xylem implied that increase of root-
sourced xylem ABA could be the primary signal in induc-
Fig. 7 Leaf chlorophyll content index (CCI) (a), total leaf N content (b) ing stomatal closure in salt-stressed potatoes. It is notable
and total leaf C content (c) of potato leaves as affected by saline irriga-
that biochar addition reduced both xylem and leaf ABA
tion and biochar treatment. B0 and B5 indicate biochar application at
concentrations which might be ascribed to decreased
the rate of 0 % and 5 % by weight in soil, respectively. S and B indicate
saline irrigations and biochar treatments, respectively. Error bars
osmotic stress due to adsorption of Na+ from the soil solu-
indicate S.E. (n = 4). tion, thereby alleviating osmotic stress to the plants. More-
over, reduced Na+ concentration, Na+/K+ ratio and
increased K+ concentration in the xylem with biochar addi-
Na+ adsorption by biochar (mg g–1)

100
a tion confirmed the reduced osmotic stress on potato due to
80 decreased Na+ in the soil solution. This effect was also
clearly exhibited by the increased midday leaf water poten-
60
tials under biochar addition (Fig. 3). Interestingly, xylem
40 b b sap K+ concentration was increased with increasing salinity
level and by biochar amendment. This increased K+ con-
20 centration with biochar amendment might be directly
c
0 related to its higher K+ contents, thereby reducing Na+
uptake, and this could be another advantage of using bio-
–20 char in reducing Na+ uptake in addition to its high Na+
0 mM NaCl 25 mM NaCl 50 mM NaCl 100 mM NaCl
adsorption potential. Whereas, on the other hand, the
Fig. 8 Na+ adsorption capacity of biochar under different salinity levels increased K+ concentration with increasing salinity levels is
and was calculated using Eqn 2 by conducting adsorption study in labo- at odds with the traditional view about salt-induced K+
ratory for 24 h. deficiency but consistent with previous studies on wheat

© 2015 Blackwell Verlag GmbH, 201 (2015) 368–378 375

Akhtar et al.
and quinoa (Cuin et al. 2009, Adolf et al. 2012). It could
be explained that plants use Na+ as a cheap osmoticum for
osmotic adjustment and maintenance of turgor pressure in
shoot, and under salinity, increased Na+ uptake has to be
accompanied by increased K+ transport to the shoot in
order to maintain suitable K+/Na+ ratio in leaves (Cuin
et al. 2009), and this might be only true under mild salt
stress condition.
Increased WUEi with biochar application further indi-
cated better plant performance under salinity stress. Con-
versely, Thomas et al. (2013) found no significant effect of
biochar on plant physiology (gs, An and WUEi) under
salinity stress. In addition, negative effect of biochar on
chlorophyll content index (CCI) was evident in the current
study which could be explained either due to reduced N
uptake because of NH4+ adsorption or due to N immobili-
zation due to higher soil C: N ratio as explained in our ear-
lier experiment (Akhtar et al. 2014) on tomato under
reduced irrigation.
Root growth was inhibited with saline irrigation because
of accumulation of Na+ ions in rhizosphere. But, addition
of biochar in the present study influences positively on root
growth (root length and root volume), which could be
explained either due to decreased Na+ contact with roots
surfaces or by reduced root resistance in biochar-amended
soil because of lowered soil bulk density. Consistent with
this, Bruun et al. (2014) also found positive effect on root
growth with biochar addition.
In conclusion, our results clearly demonstrated that
addition of biochar can significantly mitigate salinity stress
due to its high salt sorption capacity and by increasing K+
availability which ultimately improves growth, physiology
and yield of potato. Therefore, incorporation of biochar
might be a novel approach for improving crop productivity
under salinity stress. However, detailed studies should be
conducted under field conditions to evaluate the full poten-
tial of using biochar in salt-affected soils.
Acknowledgement
Financial support from Sino-Danish Center for Education
and Research (SDC) is highly acknowledged and appreci-
ated. This provided good opportunity for PhD dissertation
research. We are also thankful to Sander Brunn from
Department of Plant and Environmental Sciences, Faculty
of Science, University of Copenhagen, Denmark, for
providing the biochar material.
References
Adolf, V., S. Shabala, M. Andersen, F. Razzaghi, and S.-E. Jacob-
sen, 2012: Varietal differences of quinoa’s tolerance to saline
conditions. Plant Soil 357, 117–129.
376
Ahmad, M., A. U. Rajapaksha, J. E. Lim, M. Zhang, N. Bolan, D.
Mohan, M. Vithanage, S. S. Lee, and Y. S. Ok, 2014: Biochar
as a sorbent for contaminant management in soil and water: a
review. Chemosphere 99, 19–33.
Akhtar, S. S., G. Li, M. N. Andersen, and F. Liu, 2014: Biochar
enhances yield and quality of tomato under reduced irriga-
tion. Agric. Water Manag. 138, 37–44.
Amjad, M., J. Akhtar, M. Anwar-ul-Haq, A. Yang, S. S. Akhtar,
and S.-E. Jacobsen, 2014: Integrating role of ethylene and ABA
in tomato plants adaptation to salt stress. Sci. Hortic. 172,
109–116.
Asch, F., 2000: Determination of abscisic acid by indirect
enzyme linked immunosorbent assay (ELISA). Technical
Report. Laboratory for Agrohydrology and Bioclimatology,
Department of Agricultural Sciences, The Royal Veterinary
and Agricultural University, Taastrup, Denmark.
Atkinson, C., J. Fitzgerald, and N. Hipps, 2010: Potential
mechanisms for achieving agricultural benefits from bio-
char application to temperate soils: a review. Plant Soil
337, 1–18.
Bartell, F. E., and E. J. Miller, 1923: Adsorption by activated
sugar charcoal. J. Am. Chem. Soc. 45, 1106–1115.
Beesley, L., E. Moreno-Jimenez, J. L. Gomez-Eyles, E. Harris, B.
Robinson, and T. Sizmur, 2011: A review of biochars’ poten-
tial role in the remediation, revegetation and restoration of
contaminated soils. Environ. Pollut. 159, 3269–3282.
Bouma, T., K. Nielsen, and B. Koutstaal, 2000: Sample prepara-
tion and scanning protocol for computerised analysis of root
length and diameter. Plant Soil 218, 185–196.
Bruun, E. W., C. T. Petersen, E. Hansen, J. K. Holm, and H.
Hauggaard-Nielsen, 2014: Biochar amendment to coarse
sandy subsoil improves root growth and increases water reten-
tion. Soil Use Manag. 30, 109–118.
Chen, X., G. Chen, L. Chen, Y. Chen, J. Lehmann, M. B.
McBride, and A. G. Hay, 2011: Adsorption of copper and zinc
by biochars produced from pyrolysis of hardwood and corn
straw in aqueous solution. Bioresource Technol. 102 (19),
8877–8884.
Cheng, C.-H., J. Lehmann, J. E. Thies, and S. D. Burton, 2008:
Stability of black carbon in soils across a climatic gradient. J.
Geophys. Res. 113, G02027.
Chowdhury, M. A., A. de Neergaard, and L. S. Jensen, 2014:
Potential of aeration flow rate and bio-char addition to reduce
greenhouse gas and ammonia emissions during manure com-
posting. Chemosphere 97, 16–25.
Cuin, T. A., Y. Tian, S. A. Betts, R. Chalmandrier, and S. Shabal-
a, 2009: Ionic relations and osmotic adjustment in durum and
bread wheat under saline conditions. Funct. Plant Biol. 36,
1110–1119.
Dodd, I. C., G. Egea, and W. J. Davies, 2008: Accounting for sap
flow from different parts of the root system improves the pre-
diction of xylem ABA concentration in plants grown with het-
erogeneous soil moisture. J. Exp. Bot. 59, 4083–4093.
Doheny-Adams, T., L. Hunt, P. J. Franks, D. J. Beerling, and J.
E. Gray, 2012: Genetic manipulation of stomatal density
© 2015 Blackwell Verlag GmbH, 201 (2015) 368–378

influences stomatal size, plant growth and tolerance to
restricted water supply across a growth carbon dioxide gradi-
ent. Philos. Trans. R. Soc. Lond. B Biol. Sci. 367, 547–555.
Duan, L., D. Dietrich, C. H. Ng, P. M. Chan, R. Bhalerao, M. J.
Bennett, and J. R. Dinneny, 2013: Endodermal ABA signaling
promotes lateral root quiescence during salt stress in Arabid-
opsis seedlings. Plant Cell 25, 324–341.
Flowers, T. J., and A. L€auchli, 1983: Sodium versus potassium:
substitution and compartmentation. In: A. L€auchli, and R. L.
Bieleski, eds. Encyclopedia of Plant Physiology New Series Vol
15 B, pp. 651–681. Springer-Verlag, Berlin.
Franks, P. J., and G. D. Farquhar, 2001: The effect of exogenous
abscisic acid on stomatal development, stomatal mechanics,
and leaf gas exchange in Tradescantia virginiana. Plant Phys-
iol. 125, 935–942.
Glenn, E. P., J. J. Brown, and E. Blumwald, 1999: Salt tolerance
and crop potential of halophytes. Crit. Rev. Plant Sci. 18, 227–
255.
Hirich, A., A. Jelloul, R. Choukr-Allah, and S. E. Jacobsen, 2014:
Saline water irrigation of quinoa and chickpea: seedling rate,
stomatal conductance and yield responses. J. Agron. Crop Sci.
200, 378–389.
Ionov, M., N. Yuldasheva, N. Ulchenko, A. I. Glushenkova, and
B. Heuer, 2013: Growth, development and yield of Crambe
abyssinica under saline irrigation in the greenhouse. J. Agron.
Crop Sci. 199, 331–339.
Jeffery, S., F. G. A. Verheijen, M. van der Velde, and A. C. Bas-
tos, 2011: A quantitative review of the effects of biochar appli-
cation to soils on crop productivity using meta-analysis.
Agric. Ecosyst. Environ. 144, 175–187.
Jien, S.-H., and C.-S. Wang, 2013: Effects of biochar on soil
properties and erosion potential in a highly weathered soil.
Catena 110, 225–233.
Lashari, M. S., Y. Liu, L. Li, W. Pan, J. Fu, G. Pan, J. Zheng, J.
Zheng, X. Zhang, and X. Yu, 2013: Effects of amendment of
biochar-manure compost in conjunction with pyroligneous
solution on soil quality and wheat yield of a salt-stressed crop-
land from Central China Great Plain. Field. Crop. Res. 144,
113–118.
Lehmann, J., 2007: A handful of carbon. Nature 447, 143–144.
Lehmann, J., and S. Joseph, eds., 2009: Biochar for Environmen-
tal Management: Science and Technology. Earthscan, London.
Liang, B., J. Lehmann, D. Solomon, J. Kinyangi, J. Grossman, B.
O’Neill, J. O. Skjemstad, J. Thies, F. J. Luiz~ao, J. Petersen, and
E. G. Neves, 2006: Black carbon increases cation exchange
capacity in soils. Soil Sci. Soc. Am. J. 70, 1719–1730.
Liu, X., A. Zhang, C. Ji, S. Joseph, R. Bian, L. Li, G. Pan, and J.
Paz-Ferreiro, 2013: Biochar’s effect on crop productivity and
the dependence on experimental conditions—a meta-analysis
of literature data. Plant Soil 373, 583–594.
Lo, S. F., S. Y. Wang, M. J. Tsai, and L. D. Lin, 2012: Adsorption
capacity and removal efficiency of heavy metal ions by Moso
and Ma bamboo activated carbons. Chem. Eng. Des. 90,
1397–1406.
© 2015 Blackwell Verlag GmbH, 201 (2015) 368–378
Biochar Application under Salinity Stress
Munns, R., 2002: Comparative physiology of salt and water
stress. Plant, Cell Environ. 25, 239–250.
Munns, R., and M. Tester, 2008: Mechanisms of salinity toler-
ance. Annu. Rev. Plant Biol. 59, 651–681.
Oster, J. D., 1994: Irrigation with poor quality water. Agric.
Water Manag. 25, 271–297.
Panuccio, M. R., S. E. Jacobsen, S. S. Akhtar, and A. Muscolo,
2014: Effect of saline water irrigation on seed germination and
early seedling growth of the halophyte quinoa. AoB PLANTS
6: plu047; doi:10.1093/aobpla/plu047.
Paz-Ferreiro, J., H. Lu, S. Fu, A. Mendez, and G. Gasco, 2014:
Use of phytoremediation and biochar to remediate heavy
metal polluted soils: a review. Solid Earth 5, 65–75.
R Core Team, 2012: R: a Language and Environment for Statisti-
cal Computing. R Foundation for Statistical Computing,
Vienna, Austria. ISBN 3-900051-07-0. http://www.R-pro-
ject.org [last accessed 23 August 2013].
Razzaghi, F., S. H. Ahmadi, V. I. Adolf, C. R. Jensen, S. E.
Jacobsen, and M. N. Andersen, 2011: Water relations and
transpiration of quinoa (Chenopodium quinoa Willd.)
under salinity and soil drying. J. Agron. Crop Sci. 197,
348–360.
Regent Instruments Inc., 2009: Basic, Reg, Pro & Arabidopsis for
Root Measurement. Regent Instruments, Que´bec, QC, Can-
ada.
Samsuri, A. W., F. Sadegh-Zadeh, and B. J. Seh-Bardan, 2014:
Characterization of biochars produced from oil palm and rice
husks and their adsorption capacities for heavy metals. Int. J.
Environ. Sci. Technol. 11, 967–976.
Scheffer, F., and P. Schachtschabel, 1979: Lehrbuch der Boden-
kunde, pp. 145–187. Ferdinand Enke Verlag, Stuttgart.
Schubert, S., and A. L€auchli, 1990: Sodium exclusion mecha-
nisms at the root surface of two maize cultivars. Plant Soil
123, 205–209.
Shabala, L., A. Mackay, Y. Tian, S. E. Jacobsen, D. Zhou, and S.
Shabala, 2012: Oxidative stress protection and stomatal pat-
terning as components of salinity tolerance mechanism in qui-
noa (Chenopodium quinoa). Physiol. Plant. 146, 26–38.
Sohi, S. P., E. Krull, E. Lopez-Capel, and R. Bol, 2010: Chapter 2
– A review of biochar and its use and function in soil. Adv.
Agron. 105, 47–82.
Sun, Y., F. Yan, and F. Liu, 2013: Drying/rewetting cycles of the
soil under alternate partial root-zone drying irrigation reduce
carbon and nitrogen retention in the soil–plant systems of
potato. Agric. Water Manag. 128, 85–91.
Sun, Y., F. Liu, M. Bendevis, S. Shabala, and S. E. Jacobsen,
2014: Sensitivity of two quinoa (Chenopodium quinoa Willd.)
varieties to progressive drought stress. J. Agron. Crop Sci. 200,
12–23.
Thomas, S. C., S. Frye, N. Gale, M. Garmon, R. Launchbury, N.
Machado, S. Melamed, J. Murray, A. Petroff, and C. Winsbor-
ough, 2013: Biochar mitigates negative effects of salt additions
on two herbaceous plant species. J. Environ. Manage. 129,
62–68.
377
Akhtar et al.
Uchimiya, M., D. I. Bannon, and L. H. Wartelle, 2012a:
Retention of heavy metals by carboxyl functional groups of
biochars in small arms range soil. J. Agric. Food Chem. 60,
1798–1809.
Uchimiya, M., K. B. Cantrell, P. G. Hunt, J. M. Novak, and S.
Chang, 2012b: Retention of heavy metals in a Typic Kandiud-
ult amended with different manure-based biochars. J. Envi-
ron. Qual. 41, 1138–1149.
Wicke, B., E. Smeets, V. Dornburg, B. Vashev, T. Gaiser, W.
Turkenburg, and A. Faaij, 2011: The global technical and eco-
nomic potential of bioenergy from salt-affected soils. Energy
Environ. Sci. 4, 2669–2681.
378
Yeo, A., and T. Flowers, 1986: Salinity resistance in rice (Oryza
sativa L.) and a pyramiding approach to breeding varieties for
saline soils. Funct. Plant Biol. 13, 161–173.
Zahir, A. Z., S. S. Akhtar, A. Ahmad, Saifullah, and S. M. Na-
deem, 2012: Comparative effectiveness of Enterobacter aeroge-
nes and Pseudomonas fluorescens for mitigating the depressing
effect of brackish water on maize. Int. J. Agr. Biol. 14, 337–
344.
Zhang, X., H. Wang, L. He, K. Lu, A. Sarmah, J. Li, N. Bolan, J.
Pei, and H. Huang, 2013: Using biochar for remediation of
soils contaminated with heavy metals and organic pollutants.
Environ. Sci. Pollut. Res. 20, 8472–8483.
© 2015 Blackwell Verlag GmbH, 201 (2015) 368–378