Applied Soil Ecology 96 (2015) 114–121

Contents lists available at ScienceDirect

Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Biochar increases arbuscular mycorrhizal plant growth enhancement

and ameliorates salinity stress
Edith C. Hammera,b,c,* , Manfred Forstreutera , Matthias C. Rilliga,b , Josef Kohlera,b
a
Institute for Biology, Plant Ecology, Freie Universität Berlin, Altensteinstr. 6, D-14195 Berlin, Germany
b
Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), D-14195 Berlin, Germany
c
Institute for Biology, Microbial Ecology, Lund University, Sölvegatan 36, S-22362 Lund, Sweden

A R T I C L E I N F O A B S T R A C T

Article history: We examined combined effects of biochar, arbuscular mycorrhizal (AM) fungi and salinity on plant
Received 27 May 2015 growth and physiology to test whether and how biochar influences AM fungi mediated growth and
Received in revised form 21 July 2015 nutrition enhancements, and whether and how biochar provides amelioration in salt stressed soils. We
Accepted 23 July 2015
carried out a full three-factorial greenhouse experiment with Lactuca sativa; and a second study with a
Available online 4 August 2015
wider range of biochar and salt additions to examine physicochemical effects on soil parameters. Biochar
together with AM fungal inoculation resulted in an additional plant yield increase compared to each
Keywords:
alone under non-saline conditions. In parallel with increased plant growth, we found increased uptake of
Biochar
Black carbon
P and Mn with AM fungi and biochar addition, but to a lesser extent than biochar-induced growth
Salt stress promotion. Both factors, but especially biochar alleviated salinity-caused growth depressions, and
Mycorrhiza improved Na/K ratio in salinity stressed plants. Reduced Na uptake of plants and reduced conductivity in
Soil improvement biochar-ameliorated soils suggest that a likely mechanism involves ion adsorption to biochar surfaces.
Salt sorption Our results suggest that plants depend on symbiotic microorganisms to fully exploit biochar benefits in
soils, suggesting avenues for joint management in agriculture. Biochar may be advantageous in saline
soils, but long-term studies are required before recommendations should be given.
ã 2015 Elsevier B.V. All rights reserved.

1. Introduction
Biochar is a collective term for carbon rich soil amendments of
pyrolyzed organic material (Lehmann and Joseph, 2015). Biochars
decompose very slowly, and their addition to soils is therefore
thought to be an effective way of storing carbon to curtail the
increasing concentration of CO2 in the atmosphere (Sohi, 2013).
Despite numerous observations in both field and controlled
experiments showing that plant yield increases due to biochar
additions (Chan and Xu, 2009; Graber et al., 2010; Jeffery et al.,
2011; Lehmann et al., 2011; Van Zwieten et al., 2010), the exact
mechanistic background of biochar effects is not known. Biochars
affect soil properties differently depending on feedstock and
pyrolysis conditions, strongly differing in pH, nutrient contents
and ion exchange capacities (Lehmann and Joseph, 2015). Plant
responses to biochars may also be mediated via feedback through
biochar effects on soil microbes (Thies et al., 2015). Very little is
* Corresponding author at: Institute for Biology, Microbial Ecology, Lund
University, Sölvegatan 36, S-22362 Lund, Sweden.
E-mail addresses: Edith.Hammer@biol.lu.se, edith.hammer@gmail.com
(E.C. Hammer).
known about the interactions of biochar with root symbionts such
as arbuscular mycorrhizal (AM) fungi. AM fungi provide their host
plants with mineral nutrients and receive photosynthetically
derived carbohydrates in return (Smith and Read, 2008). The few
studies of the effects of biochar on mycorrhiza have mainly
considered root colonization and show variable results; some
researchers report root colonization levels to be strongly enhanced
by biochar (Ishii and Kadoya, 1994; Blackwell et al., 2010), whereas
others present evidence that colonization is diminished (Birk et al.,
2010; Warnock et al., 2010). Biochars, also those produced from
wood materials, typically have a large surface area and cation
exchange capacity (CEC), and therefore, its addition to soils also
increases the CEC of the soil (Joseph et al., 2009; Blackwell et al.,
2010). They can adsorb ions (Liang et al., 2006) and prevent
leaching of nutrients (Yao et al., 2011). However, those nutrients
may not necessarily be available to plants, as most roots are unable
to access the fine porous structure of the biochar due to their size,
as the finest part of the roots of most plant species are of several
hundred mm in diameter (Fitter, 2002). AM fungal hyphae are
much finer in diameter and can re-capture some of the adsorbed
nutrients and return them to their host plants (Hammer et al.,
2014). By this, a combined management of AM fungi and biochar

http://dx.doi.org/10.1016/j.apsoil.2015.07.014
0929-1393/ ã 2015 Elsevier B.V. All rights reserved.
 E.C. Hammer et al. / Applied Soil Ecology 96 (2015) 114–121
may lead to more closed nutrient cycles and more efficient
fertilizer usage. However, this has not yet been shown empirically.
Our first aim in this study is thus to examine whether possible
biochar-related enhancement of plant performance and mineral
nutrition can be further increased by the presence of AM fungi.
Our second aim is to test whether biochar ameliorates salinity
stress in plants, and whether it also may interact with AM fungi
under these conditions. It is estimated that more than 7% of the
Earth’s land is occupied by saline soils, especially in semiarid and
arid regions (Tester and Davenport, 2003), leading to major
constraints on agricultural production (Dodd and Pérez-Alfocea,
2012). AM fungi are known to ameliorate salinity stress in plants
(Evelin et al., 2009; Porcel et al., 2012) by increasing phosphorus or
general nutritional status of the plant (Giri et al., 2007), improving
water supply (Ruiz-Lozano and Azcón, 2000), and by aiding
nutrient discrimination as K+ over Na+ ions (Hajiboland et al., 2009;
Hammer et al., 2011). Little is known about the interaction of
biochars with soil salinity. It was advised to use biochar with
caution in soils as certain kinds of biochars themselves, like those
derived from poultry feedstocks, contain a high amount of salts
(Novak et al., 2009; Lehmann et al., 2011; Revell et al., 2012).
Biochar is known to sequester inorganic and organic contaminants
due to its ionic exchange, electrostatic interactions or specific
ligand binding (Ippolito et al., 2012; Xu et al., 2012). A study by
Thomas et al. (2013) found that their biochar (hardwood sawdust)
adsorbed significant amounts of added salt in a field experiment.
Thus, it might be that biochar immobilizes salt ions in moderately
saline soils, or that it may create non-saline microsites for
enhanced nutrient uptake.
We tested in a fully factorial setup the effects of biochar, AM
fungi and salt addition on growth and performance of Lactuca
sativa in a greenhouse experiment. We hypothesized that biochar
and AM fungi will interact additively on plant growth and
nutrition, i.e. that the combination of both treatments will result
in a higher plant growth than each single factor. We further
hypothesized that biochar, as AM fungi, will ameliorate salinity
stress in lettuce plants.
2. Material and methods
2.1. Soil and plant
As soil substrate we used an Albic Luvisol collected from an
experimental field of Freie Universität Berlin. The soil properties
were: sand = 69.5%, silt = 21.5% and clay = 9%; available P was
characterized as 69 mg kg 1 (calcium–acetate–lactate; Rillig et al.,
2010). Total soil N was 2 g kg 1, total soil C was 25.4 g kg 1, and pH
measured in water was 7.3 (soil:water ratio 1:5, w/v). Soil was
sieved (10 mm) to remove stones and roots. Following that, the soil
was steamed three times at 90  C (4 h, at 2-day intervals) to
eliminate AM fungi.
The plant used in the experiment was lettuce (L. sativa var.
capitata). Lettuce, being an important crop, is known to be
moderately salt sensitive (Shannon and Grieve, 1998; Kohler et al.,
2009) and is a known AM host (Azcón-Aguilar et al., 1998).
Approximately 15 days old seedlings from commercial culture
were planted in the mesocosms after washing the roots to remove
the substrate. The absence of AM fungal colonization of the
seedlings was confirmed via root staining as described in
Section 2.4.1.
2.2. AMF and biochar
The biochar was produced from pellets of coniferous wood
chips for household energy production (Olimp, Zielona Góra,
Poland), pyrolysed at 500  C for 5 h, and crushed into particles less
115
than 4 mm diameter (sieved) before mixing into the soil. Biochar
contained 0.98 mg P g 1 and 1.1 mg K g 1 (ICP-OES PerkinElmer
Optima 2100 DV, extraction see below); 85% C and 0.09% N; (Euro
EA Elemental Analyzer, HekaTech, Germany). The pH in water (1:5
soil: water ratio, w/v) was 7.6.
The AMF inoculum consisted of an AMF community originally
trap-cultured from a salt marsh (La Marina del Carmolí, Cartagena,
Región de Murcia) in Spain and was grown in trap cultures of
Sorghum bicolor. For community characterization single spores
were amplified with a nested PCR with the universal eukaryotic
primers NS1/NS4 primer pair in the first round and the SSU rRNA
gene primers NS31 and AML2 in the second PCR (Lee et al., 2008).
Purified PCR products were sequenced by LGC genomics with the
primer M13. Sequences were analyzed using BioEdit (version
7.0.9.0), showing that the inoculum was strongly dominated by
Funneliformis coronatum (Giovann.) Walker and Schuessler. The
source of inoculum had a potential infectivity of about 30 spores/g
inoculum. The inoculum consisted of a mixture of vermiculite/sand
substrate from trap cultures containing spores, hyphae, and
mycorrhizal root fragments.
2.3. Experimental design
Our greenhouse mesocosm experiment tested the effects of
biochar, AM fungi and salinity on plant performance, and a second
experiment tested physiochemical effects of biochar on soil
salinity and soil respiration with a wider range of biochar and
salt additions.
2.3.1. Mesocosm experiment
Our mesocosm experiment with lettuce had a full-factorial,
completely randomized design involving three factors with two
levels each: 0 or 5 g viable AM fungi inoculum (30 spores/g), 0 or 5%
(v/v) biochar (or 1.37% (w/w); in total 6.2 g/pot), and 0 or 4 g kg 1
soil NaCl addition. The salt addition was chosen to reach close to
the salinity threshold of 1.3 dS m 1 reported for the moderately
sensitive L. sativa in literature (Tanji and Kielen, 2002; Kohler et al.,
2009). The same amount of autoclaved inoculum was added to
non-AM plants, supplemented with a microbial wash of the
inoculum (Whatman no. 1 paper; 20 mm pore size) to provide the
microbial population except AMF. Each treatment had six
replicates, for a total of 48 pots.
Seedlings of L. sativa var. capitata were grown in 600 ml-sized
pots filled with 450 g soil. The experiment was conducted in a
greenhouse, located at Freie Universität Berlin. During the
experiment, the temperature range was 22  C during day and
16  C during night, and the relative humidity was 60%. Pots were
gravimetrically maintained around 60% water holding capacity;
soil water leaching did not occur. Plants received light during 16 h
with an intensity of 50,000 lx d 1 m 2. Salt was added as 0.5 M
NaCl solution to pots from the salt-stress treatment gradually
within the first eight days until reaching a level of 4 g NaCl kg 1
soil. The equivalent in deionized water was added to pots from the
non-stressed treatment. Two plants belonging to the treatment
salt and AM fungi addition died early during the experiment and
were not considered in any analysis. After 35 days, pots were
destructively harvested.
2.3.2. Soil property experiment
In a second experiment we incubated the same soil without
plants with a fully factorial design involving the factors biochar and
salt addition at 3 levels each: 0, 5 or 15% (v/v) biochar and 0, 4 or
10 g kg 1 NaCl addition. The soil was kept in 200 ml screw lid cups
at room temperature with loosely closed lids for 40 days, and
regular gravimetrical water addition ensured maintenance of
moisture. We measured conductivity, pH after the incubation, as
116 E.C. Hammer et al. / Applied Soil Ecology 96 (2015) 114–121

described above. We determined water holding capacity of the

soils, and loss of ignition to assess soil organic carbon at a
temperature of 500  C.

2.4. Analyses

2.4.1. Plant
During the growth period we measured stomatal conductance
and photosynthesis rate (net assimilation) on one fresh leaf of the
youngest part of the plant for each pot after two weeks with the
portable gas exchange instrument HCM-1000 (Heinz Walz GmbH,
Effeltrich, Germany) as described in the supplementary data.
After harvest we determined root and shoot dry weight
following drying at 60  C for 48 h. Plant tissues were ground
before chemical analysis of nutrients. The concentrations of P, Na,
K, Mg, Ca, Fe and Mn in plant shoot and biochar material were
determined after digestion in nitric–perchloric acid (5:3, v/v) for
6 h and quantified using an ICP-OES (PerkinElmer Optima 2100
DV). For analysis of nitrogen and carbon concentration, ground
shoot samples were weighed into tin capsules and analyzed by an
Elemental Analyser (EuroEA, HekaTech, Germany) with acetanilide
(Merck, Darmstadt, Germany) as internal standard. Additional data
on free proline concentration indicating plant stress levels are
described in the supplementary data.
Root colonization by AMF was determined by clearing with 10%
KOH and staining with 0.05% trypan blue (Phillips and Hayman,
1970) and the percentage of AMF root colonization was determined
using the gridline intersect method (McGonigle et al., 1990).

2.4.2. Soil
We measured pH and conductivity in a 1:5 extract (soil:water,
w/v) at start and after the end of both experiments using a
standard pH-meter (Knick 761 calimatic, Berlin) and a conductivity
meter (seven easy LF, Mettler Toledo, Gießen, Germany), respec-
tively.
Nitrogen and carbon concentrations of the soils were measured
as described above. We examined biochar particles obtained from
soil of the biochar x AM fungi treatment for hyphal colonization
with a Leica M165C microscope. Fig. 1. Biomass of L. sativa. Effect of the biochar (B), inoculation with arbuscular
mycorrhizal fungi (A) and salt stress (S) on shoot (a) and root (b) dry weight of L.
We measured bacterial activity as a contributor to soil
sativa (n = 6) (+ P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001; only significant factors or
respiration described in the Supplementary data. significant interaction terms are shown). Photograph of two representative pots of
each treatment (c). A line in the legend denotes the presence of the respective factor.
2.5. Statistical analysis

Treatment effects were tested in R version 2.15.1 (R Core Team,
2013) using a general linear model (Zuur et al., 2009) verifying
normal distribution of residuals using Shapiro-test. Homogeneity
of variance was tested using the Levene’s-test. In the cases where
the residuals were not normally distributed we used log-trans-
formations (which were: root dry weight, conductivity, bacterial
growth, N, P, Fe, Mg). Nutrient data was analyzed for treatment
groupings and correlations of tissue nutrient concentrations with a
principal component analysis (PCA) performed in JMP Pro 11. (SAS
Institute Inc., 2013). All parameters of the mesocosm experiment
were analyzed using three-way ANOVA, and those of the soil
property experiment using a two-way ANOVA.
3. Results
3.1. Mesocosm experiment
Both the addition of biochar and inoculation with AM fungi
significantly increased shoot biomass of lettuce plants (Fig. 1a).
Under the influence of both, plants showed highest yields, reaching
more than double the size compared to control plants. Both
biochar and AM fungi also increased plant biomass under salinity
stress, which generally had negative effects on plant growth and
physiology. Root biomass was increased by biochar, especially
under non-saline conditions, while AM fungi had no effect (Fig. 1b).
Biochar alleviated salinity induced reduction in net assimilation
rates (P < 0.05), but neither AM fungi nor biochar influenced
stomatal conductance (Fig. S1).
Both the addition of biochar and AM fungi increased uptake
(total shoot content) of the nutrients P and Mn, both under non-
saline and under saline conditions, but no significant additive
effect could be found (Fig. 2a and b). N uptake in lettuce was
unchanged under biochar addition, even though biomass doubled
in those plants. This led to a low shoot N concentration (below 1.5%,
Table 1), indicating N limitation in biochar amended plants.
Soil salinity led to decreased shoot tissue concentrations of the
base cations Ca and Mg in parallel with increased Na concen-
trations (Table 1; Fig. 2c), K concentration was halved in salinity
stressed plants (Fig. 2d). Also biochar addition generally reduced
base cation concentrations in non-salt stressed plants, (Fig. 2c and
d, Table 1). Under salinity, AM fungi and especially biochar
attenuated excessive Na concentrations, and increased K nutrition.
Biochar addition almost doubled the tissue K/Na ratio of salt
stressed plants (Table 1), and also AM fungi increased the K/Na
 E.C. Hammer et al. / Applied Soil Ecology 96 (2015) 114–121 117

Fig. 2. Shoot tissue nutrients. Effect of the biochar (B), inoculation with arbuscular mycorrhizal fungi (A) and salt stress (S) on P (a), Mn (b), Na (c), and K (d) concentrations of
L. sativa leaves after harvest (n = 6) (+ P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001; only significant factors or significant interaction terms are shown). A line in the legend
denotes the presence of the respective factor. A principle component analysis visualizes changes in nutrient concentrations in L. sativa shoots according to the treatments (e).
Left: score plot. Open symbols: salt addition; solid symbols: no salt addition; blue square symbols: biochar addition; rectangular vertical yellow symbols: AM fungi
inoculation; circular green symbols: biochar and AMF addition; rectangular horizontal grey symbols: control or saline control. Right: loading plot. Shoot tissue concentrations
of N, P, K, Na and other micronutrients. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

ratio of salt stressed plants by 50% (Table 1), but no additive effect
with biochar occurred. Salinity clearly separated the dataset of
shoot tissue nutrients in a principal component analysis into two
main groups, with Na concentrations as the most important
loading variable (Fig. 2e, salinity treatment clustering in the upper
left). A further grouping was determined by biochar addition under
non-saline conditions, those samples being both lower in
concentrations of N, P and autocorrelated micronutrients, pre-
sumably due to dilution in a higher biomass (Fig. 2e, biochar
treated samples clustering in the lower left).
118 E.C. Hammer et al. / Applied Soil Ecology 96 (2015) 114–121

Table 1
Effect of inoculation with arbuscular mycorrhizal fungi (AMF) and amendment of biochar on nutrients concentrations of shoots of L. sativa seedlings grown at two levels of
salinity (mean  SE; n = 6). F values of a three-factor-ANOVA are shown below (+ P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001)
1 1 1
AMF Bio-char Salt N (%) Fe (mg g ) Ca (mg g ) Mg (mgg ) K/Na ratio

2.2  0.3 0.28  0.0 33  1 2.50  0.1 10.6  0.8

+ 2.5  0.3 0.32  0.0 29  1 2.10  0.1 6.9  1.3
+ 1.4  0.1 0.11  0.0 24  1 1.61  0.1 12.4  1.1
+ + 1.4  0.1 0.12  0.0 25  1 1.73  0.1 11.5  0.54
+ 1.8  0.1 0.27  0.1 28  2 1.55  0.1 0.29  0.03
+ + 1.7  0.2 0.10  0.0 24  1 1.55  0.1 0.43  0.03
+ + 1.5  0.0 0.08  0.0 25  1 1.78  0.1 0.57  0.05
+ + + 1.5  0.0 0.07  0.0 22  1 1.68  0.1 0.56  0.03

F-values
AMF NS NS 5.3* NS NS
Biochar 33*** 36*** 19*** 15*** 68***
Salt NS 5.44* 7* 29*** 2630***
AMF:biochar NS NS NS NS NS
Biochar:salt 7.3* NS NS 46*** 6.7*
AMF:salt NS 3.4 NS NS 7.3*
AMF:biochar:salt NS 5.8* NS 5.6* 5.8*

No changes could be detected in soil bacterial activity after AM 4. Discussion

fungal inoculation or biochar amendment both under saline and
non-saline conditions (Fig. S3). AM fungal extraradical hyphae
colonized biochar particles in the soils of the experiment (Fig. S4).
Salt addition increased soil conductivity more than 10-fold (
P < 0.001, Table 2). Biochar led to a moderate but highly significant
decrease of conductivity (P < 0.001). Soil pH was slightly lowered
after addition of biochar (P < 0.001), while salt addition increased
it (P < 0.001). AM fungi and AM fungi together with biochar
diminished this effect (P < 0.05, Table 2). However, pH was always
very close to neutral, so that changes in hydronium ion activity
were minor. Soil C content was increased by biochar amendments
compared to control pots (P < 0.01, Table 2). A root length of
approx. 21% was colonized by AM fungi after inoculation in non-
saline systems, which tended to decrease with elevated salinity to
16% (Table 2).
3.2. Soil property experiment
When testing a wider concentration range of biochar and
salinity on soil properties, we confirmed that soil conductivity was
clearly though moderately diminished by increasing biochar
addition (P < 0.05; Fig. 3a). Our biochar had no effect on pH or
water holding capacity, while both significantly decreased with
increasing salinity (Fig. 3b and c).
Biochar enhanced plant growth in our experiment, as docu-
mented in literature (e.g. Chan and Xu, 2009; Graber et al., 2010),
and as expected as we were using a rather sandy and nutrient poor
soil (Jeffery et al., 2011). Often, changes in pH are suggested to
cause changes in nutrient availability of the soil that lead to
increased plant growth (Lehmann, 2007), or a changed water
retention capacity of the soil (Glaser et al., 2002; Basso et al., 2013).
In contrast, we found no or hardly any change in water retention
capacity or pH after biochar addition in our study. Instead, we
found an increase in plant nutrient contents, especially an increase
in P and Mn plant content, suggesting that a better P and/or Mn
nutrition have contributed to the observed increase in plant
growth. However, the magnitude of increased nutrition was not
equivalent to but lower than the observed growth enhancements.
The amount of mineral nutrients added via the biochar to the
system was low, as the woody parent material consisted mainly of
carbon structures. The 6.2 g biochar added to each mesocosm
(which are 5%, v/v) contained 5.5 mg N und 6 mg total P, compared
to 540 mg N and 31 mg plant available P in the soil of each pot. A
rather long pyrolysis at 500  C, as in our case, converts the vast
majority of carbon compounds in organic material into amorphous
structures (Downie et al., 2009). Thus, a fertilization effect (as e.g.
suggested in Jones et al., 2012) with both mineral nutrients or

Table 2
Effect of inoculation with arbuscular mycorrhizal fungi (AMF) and amendment of biochar on root colonization, electric conductivity, pH, soil C and C/N ratio of rhizosphere soil
of L. sativa seedlings grown at two levels of salinity (mean  SE; n = 6). F values of a three-factor-ANOVA are shown below (+ P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001)

AMF Biochar Salt Root colonization (%) Conductivity (mS cm 1

) pH Soil C (%) C/N ratio

1.3  0.2 109  2 7.3  0.04 2.0  0.52 12.2  0.24

+ 21  1.4 142  27 7.5  0.02 2.1  0.38 12.9  0.36
+ 1.2  0.4 130  5 7.2  0.01 3.7  0.47 21.7  1.46
+ + 21  2.0 134  2 7.2  0.02 4.5  0.63 21.6  2.98
+ 0.00  0.0 1280  55 7.5  0.02 2.0  0.28 13.8  0.8
+ + 17  1.5 1450  220 7.5  0.02 2.0  0.19 11.9  0.17
+ + 0.67  0.26 1130  54 7.4  0.02 3.5  0.85 27.0  4.79
+ + + 15  1.6 1170  95 7.4  0.03 3.3  0.49 17.6  1.61

F-values
Biochar NS 3.4+ 74*** 11** 107***
AMF 92*** NS NS NS NS
Salt NS 363*** 55*** NS NS
Biochar:AMF NS NS NS NS NS
Biochar:salt NS 4.7* NS NS NS
AMF:salt NS NS 12** NS NS
Biochar:AMF:salt NS NS 4.2* NS NS
 E.C. Hammer et al. / Applied Soil Ecology 96 (2015) 114–121
Fig. 3. Soil parameters. Effect of biochar and salinity at three addition levels on electric conductivity (a), pH (b), water holding capacity (c) and soil respiration (d) in bare soil at
the end of the incubation time (n = 6) (+ P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001; only significant factors or significant interaction terms are shown). (+) denotes 4 g salt
addition per kg soil, or 5% (vol) biochar addition, respectively, (++) denotes 10 g salt addition per kg soil, or 15% (vol) biochar addition, respectively.
labile carbon compounds is unlikely to be a major explaination of
our results. This reasoning is supported by an unchanged bacterial
activity in the soil, which tends to increase and quickly respond to
changes in availability of labile carbon (Rousk et al., 2013) that may
be included in biochars. Biochar can affect nutrient release,
retention or immobilization (Blackwell et al., 2010) also by its
surface properties, e.g. through its cation exchange capacity and
anion exchange capacity. The increased nutrient uptake of plants
grown in biochar-amended soils indicates a better nutrient
availability. Biochar could positively influence plant nutrition in
several ways: sorption of nutrients, especially to the inner surface
parts of biochar particles, could prevent their fixation onto soil
colloids, by which nutrients are easily immobilized. Biochars can
be very efficient agents for phosphate removal from wastewaters
(Yao et al., 2011; Yao et al., 2013) and soil leachates (Laird et al.,
2010). These nutrient ions are at least partly conserved for nutrient
cycling, as biochar can increase the P availability of soil (Lashari
et al., 2013). However, most nutrients were found in lowered tissue
concentration past biochar addition in accordance with a greater
plant body, indicating that growth may finally be nutrient limited.
In an agricultural system, simultaneous moderate fertilization may
overcome this problem.
We found an additive effect between AM fungi and biochar on
plant shoot biomass. AM fungal inoculation increased biomass and
especially P uptake of the plant. Improved P nutrition via AM fungi
and subsequent increased plant growth is one of the most
important factors in the mycorrhizal symbiosis (Smith and Read,
2008). Recently, two smaller studies dealt with interactive effects
of biochar and AM fungi on plant growth, one field- and one
greenhouse study. While the field study from South Aftica could
not depict any biochar effects on shoot yields of tomato plants
(Nzanza et al., 2012), the greenhouse study from Indonisia found a
strong synergistic effect of biochar and AM fungi on growth and
nutrient uptake of tree seedlings (Budi and Setyaningsih, 2013).
However, little is known about the mechanistic background so far.
Hammer et al. (2014) found that AM fungi transfer biochar-surface
adsorbed phosphate to the host plant. This additional P pool is
largely not available to roots, as a great part of the surface structure
of a woody biochar consists of pore space of a diameter less than
that of roots or root hairs (Fitter, 2002). AM fungal hyphae, by
contrast, are both thin and long enough to permit exploration of
this pore space (Fig. S4; Hammer et al., 2014). If biochar can
maintain nutrients in the ecosystem, and if AM fungi can access
and recycle those, leaching or fixation is prevented and nutrient
cycles will become more closed. This mechanism of biochar as a
nutrient provider and AM fungi as the uptake mediator could
119
explain the additive effect of biochar and AMF amendment on plant
growth. However, in this study we did not find any additive effects
in plant tissue nutrients due to AM fungi and biochar addition,
indicating that different mechanism than nutrition only is needed
to explain the additive effect of AM fungi and biochar on plant
yield, which needs further investigation into plant physiology,
microbial communities or detailed soil parameters.
Salinity is a frequent problem in agriculturally used soils,
limiting crop production, and is a major cause of abandonment of
lands (Dodd and Pérez-Alfocea, 2012). Salt in the soil solution
generates a lowered water potential, which leads to physiological
drought for the plant (Munns, 2002; Mahajan and Tuteja, 2005). A
high concentration of the water soluble sodium (Na) and chloride
(Cl) ions leads to increased uptake of these and by this to toxic
damage in cells, generates osmotic stress in plants and hinders the
absorption of essential nutrients as Ca and K (Pérez-Alfocea et al.,
1993; Munns et al., 2006).
The potential of biochar to abate salt stress in plants is still
under-researched. To our knowledge, there are only very few
recent studies examining the effect of biochar on plant growth
under salinity (Lashari et al., 2013; Thomas et al., 2013; Lashari
et al., 2015). Lashari et al. (2013, 2015) used a biochar manure
compost, i.e. a mixture of pyrolysed carbon and fresh organic
matter, which ameliorated salt stress, but the mechanisms behind
this remained unsolved. Thomas et al. (2013) added salt over a top
dressing of biochar to forb pot cultures and concluded that biochar
ameliorated salt stress through salt sorption, as biochar material
showed strongly increased conductivity following salt addition.
We also found a clear amelioration of salt stress in plants after
biochar amendment in combination with reduced salt strength in
both plants and soil (Fig. 2c, Table 1). This could be caused by an
indirect dilution effect in generally bigger biochar-amended
plants, but biochar could also be the direct cause for the lower
Na concentrations in the plant cells that in concequence allowed
for a higher plant growth. The latter argument finds support as we
found a decreased conductivity in salinized soil after biochar
addition, likely caused by Na immobilization from the soil solution
onto the biochar surface (Table 1, Fig. 3). The reduction of
conductivity by biochar was particularly visible in Expt. 2. This
effect was measured at the end of the incubation time of three
months, and biochars typically run through an aging and oxidation
process (Cheng et al., 2006), which increases their cation exchange
capacity (Liang et al., 2006; Cheng et al., 2008). However, even
though this reduction in conductivity was highly significant, the
effect size measured appears too small to fully explain the strong
biochar amelioration of salinity induced plant growth in Expt. 1. A
120 E.C. Hammer et al. / Applied Soil Ecology 96 (2015) 114–121
generally better plant nutrition status may help to overcome
salinity stress. Not only Na levels were reduced, but also K and Mg
concentrations and total K and Mg uptake (Table 1) were clearly
increased in plants that received biochar and/or AM fungi addition.
High level of Na in the growth environment of a plant interferes
with the uptake of essential nutrients similar to Na, especially K.
Hammer et al. (2011) found that AM fungi under salinity have a
strongly increased tissue K/Na ratio compared to their growth
environment, which can help to maintain favorable K/Na ratios in
their host plants. Our results indicate that also biochar improves
selective nutrient ion uptake under salinity. Also here, a pure
fertilization effect is unlikely: the biochar that we added contained
6.8 mg of K per pot, but even if this K was fully available for uptake
it is not sufficient to explain the increase of additional 24 mg K
uptake by biochar-amended plants under salinity. Biochar can
retain significant amounts of cations on its surface (Liang et al.,
2006; Chan and Xu, 2009), and surface affinities to Na may differ
comparted than to K ions.
While both AM fungi and biochar addition ameliorated
salinity stress in L. sativa, we did not find any additive effect
between those under salinity. Also in other parameters
measured, such as stress indicators and nutrition, biochar had
a stronger ameliorating effect than AM fungi under salinity. An
earlier study found that biochar led to increased plant drought
resistance (Mulcahy et al., 2013). Considering these results,
biochar may be a promising tool to ameliorate saline agricultural
soils. However, the long-term effects of biochar in saline soils
need to be assessed prior to field applications. Our experiment
measured effects during a change in the environment, as both
salt and biochar were freshly added to our mesocosms. The
alleviating effect of biochar under salinity may decrease with
time, when its CEC-sites equilibrate.
The contrasting minor changes in pH observed in Expts. 1 and 2
likely derive from changes in root growth. Plants were present only
in Expt. 1, where biochar increased and salt addition decreased root
penetration and thus likely root exudates, leading to a relatively
decreased and increased pH, vice versa.
In conclusion, we found that biochar strongly alleviated, at least
during short term, salinity stress in lettuce plants, presumably
explained by ion soption to biochar surfaces. Our results indicate
that improved nutrition may be an important explanatory variable
of biochar effects, and that plants depend on symbiotic micro-
organisms to fully exploit the potential of biochar benefits in soils.
The additive effect on plant growth suggests avenues for a
profitable joint management of AM fungi and biochar application
in agriculture.
Acknowledgements
EH was supported by a FORMAS and a Marie Curie grant (FP7-
PEOPLE-2010-IEF NANOSOIL) and JK by a Marie Curie grant (FP7-
PEOPLE-2010-IEF-MYCOCARBOSE). We thank the anonymous
reviewers for helpful comments and suggestions. We would also
like to thank Ulrike Hemberger and Nadine Reinhardt for technical
assistance, and Philipp Hoelzmann for measuring nutrients with
ICP-OES.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
apsoil.2015.07.014.
References
Azcón-Aguilar, R., Handley, L.L., Scrimgeour, C.M., 1998. The delta N-15 of lettuce
and barley are affected by AM status and external concentration of N. New
Phytol. 138, 19–26.
Basso, A.S., Miguez, F.E., Laird, D.A., Horton, R., Westgate, M., 2013. Assessing
potential of biochar for increasing water-holding capacity of sandy soils. Glob.
Change Biol. Bioenergy 5, 132–143.
Birk, J.J., Steiner, C., Teixeira, W.G., Zech, W., Glaser, B., 2010. Microbial response to
charcoal amendments and fertilization of a highly weathered tropical soil. In:
Woods, W.I., Teixeira, W.G., Lehmann, J., Steiner, C., WinklerPrins, A., Rebellato,
L. (Eds.), Amazonian Dark Earths: Wim Sombroek’s Vision. Springer, Berlin, pp.
309–324.
Blackwell, P., Krull, E., Butler, G., Herbert, A., Solaiman, Z., 2010. Effect of banded
biochar on dryland wheat production and fertiliser use in south-western
Australia: an agronomic and economic perspective. Aust. J. Soil Res. 48,
531–545.
Budi, S.W., Setyaningsih, L., 2013. Arbuscular mycorrhizal fungi and biochar
improved early growth of neem (Melia azedrach Linn.) seedling under
greenhouse conditions. J. Manaj. Hutan Trop. 19 (2), 103–110.
Chan, K.Y., Xu, Z., 2009. Biochar: nutrient properties and their enhancement. In:
Lehmann, J., Joseph, S. (Eds.), Biochar for Environmental Management: Science
and Technology. Earthscan, London, pp. 67–84.
Cheng, H., Lehmann, J., Thies, J.E., Burton, S.D., Engelhard, M.H., 2006. Oxidation of
black carbon by biotic and abiotic processes. Org. Geochem. 37, 1477–1488.
Cheng, C.H., Lehmann, J., Engelhard, M., 2008. Natural oxidation of black carbon in
soils: changes in molecular form and surface charge along a climosequence.
Geochim. Cosmochim. Acta 72, 1598–1610.
Dodd, I., Pérez-Alfocea, F., 2012. Microbial amelioration of crop salinity stress. J. Exp.
Bot. 63, 3415–3428.
Downie, A., Crosky, A., Munroe, P., 2009. Physical properties of biochar. In: Lehmann,
J., Joseph, S. (Eds.), Biochar for Environmental Management: Science and
Technology. Earthscan, London, pp. 13–32.
Evelin, H., Kapoor, R., Giri, B., 2009. Arbuscular mycorrhizal fungi in alleviation of
salt stress: a review. Ann. Bot. 104, 1263–1280.
Fitter, A., 2002. Characteristics and functions of root systems, In: Waisel, Y., Eshel, A.,
Kafkafi, U. (Eds.), Plant Roots: The Hidden Half. 3rd edition Dekker Inc., NY, USA.
Giri, B., Kapoor, R., Mukerji, K.G., 2007. Improved tolerance of Acacia nilotica to salt
stress by arbuscular mycorrhiza Glomus fasciculatum, may be partly related to
elevated K+/Na+ ratios in root and shoot tissues. Microb. Ecol. 54, 753–760.
Glaser, B., Lehmann, J., Zech, W., 2002. Ameliorating physical and chemical
properties of highly weathered soils in the tropics with charcoal—a review. Biol.
Fertil. Soils 35, 219–230.
Graber, E.R., Hare, Y.M., Kolton, M., Cytryn, E., Silber, A., David, D.R., Tsechansky, L.,
Borenshtein, M., Elad, Y., 2010. Biochar impact on development and productivity
of pepper and tomato grown in fertigated soilless media. Plant Soil 337,
481–496.
Hajiboland, R., Joudmand, A., Fotouhi, K., 2009. Mild salinity improves sugar beet
(Beta vulgaris L.) quality. Acta Agric. Scand. B-S P 59, 295–305.
Hammer, E., Balogh-Brunstad, Z., Jakobsen, I., Olsson, P.A., Stipp, S.L.S., Rillig, M.C.,
2014. A mycorrhizal fungus grows on biochar and captures phosphorus from its
surfaces. Soil Biol. Biochem. 77, 252–260.
Hammer, E.C., Nasr, H., Pallon, J., Olsson, P.A., Wallander, H., 2011. Elemental
composition of arbuscular mycorrhizal fungi at high salinity. Mycorrhiza 21,
117–129.
Ippolito, J.A., Laird, D.A., Busscher, W.J., 2012. Environmental benefits of biochar. J.
Environ. Qual. 4, 967–972.
Ishii, T., Kadoya, K., 1994. Effects of charcoal as a soil conditioner on citrus growth
and vesicular–arbuscular mycorrhizal development. J. Jpn. Soc. Hortic. Sci. 63,
529–535.
Jeffery, S., Verheijen, F.G.A., van der Velde, M., Bastos, A.C., 2011. A quantitative
review of the effects of biochar application to soils on crop productivity using
meta-analysis. Agric. Ecosyst. Environ. 144, 175–187.
Jones, D.L., Rousk, J., Edwards-Jones, G., DeLuca, T.H., Murphy, D.V., 2012. Biochar-
mediated changes in soil quality and plant growth in a three year field trial. Soil
Biol. Biochem. 45, 113–124.
Joseph, S., Peacocke, C., Lehmann, J., Munroe, P., 2009. Developing a biochar
classification and test methods. In: Lehmann, J., Joseph, S. (Eds.), Biochar for
Environmental Management: Science and Technology. Earthscan, London, pp.
101–126.
Kohler, J., Hernández, J., Caravaca, F., Roldán, A., 2009. Induction of antioxidant
enzymes is involved in the greater effectiveness of a PGPR versus AM fungi with
respect to increasing the tolerance of lettuce to severe salt stress. Environ. Exp.
Bot. 65, 245–252.
Laird, D., Fleming, P., Wang, B.Q., Horton, R., Karlen, D., 2010. Biochar impact on
nutrient leaching from a Midwestern agricultural soil. Geoderma 158, 436–442.
Lashari, M.S., Liu, Y., Li, L., Pan, W., Fu, J., Pan, G., Zheng, J., Zheng, J., Zhang, X., Yu, X.,
2013. Effects of amendment of biochar-manure compost in conjunction with
pyroligneous solution on soil quality and wheat yield of a salt-stressed cropland
from Central China Great Plain. Field Crop Res. 144, 113–118.
Lashari, M.S., Ye, Y., Ji, H., Li, L., Kibue, G.W., Lu, H., Zheng, J., Pan, G., 2015. Biochar–
manure compost in conjunction with pyroligneous solution alleviated salt
stress and improved leaf bioactivity of maize in a saline soil from central China:
a 2-year field experiment. J. Sci. Food Agric. 95 (5), 1321–1327.
 E.C. Hammer et al. / Applied Soil Ecology 96 (2015) 114–121
Lee, J., Lee, S., Young, J.P.W., 2008. Improved PCR primers for the detection and
identification of arbuscular mycorrhizal fungi. FEMS Microbiol. Ecol. 65, 339–
349.
Lehmann, J., 2007. Bio-energy in the black. Front. Ecol. Environ. 5, 1–387.
Lehmann, J., Joseph, S., 2015. Biochar for environmental management: an
introduction. In: Lehmann, J., Joseph, S. (Eds.), Biochar for Environmental
Management: Science and Technology. Earthscan, London, pp. 1–14.
Lehmann, J., Rillig, M.C., Thies, J., Masiello, C.A., Hockaday, W.C., Crowley, D., 2011.
Biochar effects on soil biota—a review. Soil Biol. Biochem. 43, 1812–1836.
Liang, B., Lehmann, J., Solomon, D., Kinyangi, J., Grossman, J., O’Neill, B., Skjemstad, J.
O., Thies, J., Luizão, F.J., Petersen, J., Neves, E.G., 2006. Black carbon
increases cation exchange capacity in soils. Soil Sci. Soc. Am. J. 70,
1719–1730.
Mahajan, S., Tuteja, N., 2005. Cold, salinity and drought stresses: an overview. Arch.
Biochem. Biophys. 444, 139–158.
McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990. A new
method which gives an objective measure of colonization of roots by vesicular–
arbuscular mycorrhizal fungi. New Phytol. 115, 495–501.
Mulcahy, D.N., Mulcahy, D.L., Dietz, D., 2013. Biochar soil amendment increases
tomato seedling resistance to drought in sandy soils. J. Arid Environ. 88,
222–225.
Munns, R., 2002. Comparative physiology of salt and water stress. Plant Cell Environ.
25, 239–250.
Munns, R., James, R.A., Läuchli, A., 2006. Approaches to increasing the salt tolerance
of wheat and other cereals. J. Exp. Bot. 57, 1025–1043.
Nzanza, B., Marais, D., Soundy, P., 2012. Effect of arbuscular mycorrhizal fungal
inoculation and biochar amendment on growth and yield of tomato. Int. J. Agric.
Biol. 14, 965–969.
Novak, J.M., Lima, I., Xing, B., Gaskin, J.W., Steiner, C., Das, K.C., Ahmedna, M., Rehrah,
D., Watts, D.W., Busscher, W.J., Schomberg, H., 2009. Characterization of
designer biochar produced at different temperatures and their effects on a
loamy sand. Ann. Environ. Sci. 3, 195–206.
Pérez-Alfocea, F., Estañ, M.T., Caro, M., Guerrier, G., 1993. Osmotic adjustment in
Lycopersicon esculentum and L. pennellii under NaCl and polyethylene glycol
6000 iso-osmotic stresses. Physiol. Plant. 87, 493–498.
Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and
staining parasitic and vesicular–arbuscular mycorrhizal fungi for rapid
assessment of infection. Trans. Br. Mycol. Soc. 55, 158–161.
Porcel, R., Aroca, R., Ruiz-Lozano, J.M., 2012. Salinity stress alleviation using
arbuscular mycorrhizal fungi. A review. Agron. Sustain. Dev. 32, 181–200.
R Core Team, 2013. R: A language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0,
http://www.R-project.org/.
Revell, K.T., Maguire, R.O., Agblevor, F.A., 2012. Influence of poultry pitter biochar on
soil properties and plant growth. Soil Sci. 177, 402–408.
121
Rillig, M.C., Wagner, M., Salem, M., Antunes, P.M., George, C., Ramke, H.G., Titirici, M.
M., Antonietti, M., 2010. Material derived from hydrothermal carbonization:
effects on plant growth and arbuscular mycorrhiza. Appl. Soil Ecol. 45, 238–242.
Rousk, J., Dempster, D.N., Jones, D.L., 2013. Transient biochar effects on decomposer
microbial growth rates: evidence from two agricultural case-studies. Eur. J. Soil
Sci. 64, 770–776.
Ruiz-Lozano, J.M., Azcón, R., 2000. Symbiotic efficiency and infectivity of an
autochthonous arbuscular mycorrhizal Glomus sp from saline soils and Glomus
deserticola under salinity. Mycorrhiza 10, 137–143.
SAS Institute Inc, 2013. JMP Pro 11.2.0 statistical software. Cary, NC, USA.
Shannon, M.C., Grieve, C.M., 1998. Tolerance of vegetable crops to salinity. Sci.
Hortic. Amst. 78, 5–38.
Sohi, S.P., 2013. Pyrolysis bioenergy with biochar production—greater carbon
abatement and benefits to soil. Glob. Change Biol. Bioenergy 5, 1–3.
Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis. Academic Press, Cambridge.
Tanji, K.K., Kielen, N.C., 2002. Agricultural Drainage Water Management in Arid and
Semi-Arid Areas. Irrigation and Drainage Paper 61. Food and Agriculture
Organization of the United Nations (FAO), Rome.
Tester, M., Davenport, R., 2003. Na+ tolerance and Na+ transport in higher plants.
Ann. Bot. Lond. 91, 503–527.
Thies, J.E., Rillig, M.C., Graber, E.R., 2015. Biochar effects on the abundance, activity
and diversity of the soil biota. In: Lehmann, J., Joseph, S. (Eds.), Biochar for
Environmental Management: Science and Technology. Earthscan, London, pp.
327–390.
Thomas, S.C., Fry, S., Gale, N., Garmon, M., Launchbury, R., Machado, N., Melamed, S.,
Murray, J., Petroff, A., Winsborough, C., 2013. Biochar mitigates negative effects
of salt additions on two herbaceous plant species. J. Environ. Manag. 129.
Van Zwieten, L., Kimber, S., Morris, S., Chan, K.Y., Downie, A., Rust, J., Joseph, S.,
Cowie, A., 2010. Effects of biochar from slow pyrolysis of papermill waste on
agronomic performance and soil fertility. Plant Soil 327, 235–246.
Warnock, D.D., Mummey, D.L., McBride, B., Major, J., Lehmann, J., Rillig, M.C., 2010.
Influences of non-herbaceous biochar on arbuscular mycorrhizal fungal
abundances in roots and soils: results from growth-chamber and field
experiments. Appl. Soil Ecol. 46, 450–456.
Xu, G., Lv, Y., Sun, J., Shao, H., Wei, L., 2012. Recent advances in biochar applications
in agricultural soils: benefits and environmental implications. Clean Soil Air
Water 40, 1093–1098.
Yao, Y., Gao, B., Inyang, M., Zimmerman, A.R., Cao, X.D., Pullammanappallil, P., Yang,
L.Y., 2011. Removal of phosphate from aqueous solution by biochar derived from
anaerobically digested sugar beet tailings. J. Hazard. Mater. 190, 501–507.
Yao, Y., Gao, B., Chen, J.J., Yang, L.Y., 2013. Engineered biochar reclaiming phosphate
from aqueous solutions: mechanisms and potential application as a slow-
release fertilizer. Environ. Sci. Technol. 47, 8700–8708.
Zuur, A.F., Ieno, E.N., Walker, N.J., Saveliev, A.A., Smith, G.M., 2009. Mixed Effects
Models and Extensions in Ecology with R. Springer-Verlag, New York.