Plant Soil (2010) 337:481–496
DOI 10.1007/s11104-010-0544-6
REGULAR ARTICLE
Biochar impact on development and productivity of pepper
and tomato grown in fertigated soilless media
Ellen R. Graber & Yael Meller Harel & Max Kolton & Eddie Cytryn & Avner Silber &
Dalia Rav David & Ludmilla Tsechansky & Menahem Borenshtein & Yigal Elad
Received: 15 June 2010 / Accepted: 17 August 2010 / Published online: 2 September 2010
# Springer Science+Business Media B.V. 2010
Abstract The impact of additions (1–5% by weight)
of a nutrient-poor, wood-derived biochar on pepper
(Capsicum annuum L.) and tomato (Lycopersicum
esculentum Mill.) plant development and productivity
in a coconut fiber:tuff growing mix under optimal
fertigation conditions was examined. Pepper plant
development in the biochar-treated pots was signifi-
cantly enhanced as compared with the unamended
controls. This was reflected by a system-wide
increase in most measured plant parameters: leaf area,
canopy dry weight, number of nodes, and yields of
buds, flowers and fruit. In addition to the observed
Responsible Editor: Jorge Vivanco.
E. R. Graber (*) : M. Kolton : E. Cytryn : A. Silber :
L. Tsechansky
Institute of Soil, Water and Environmental Sciences,
The Volcani Center, Agricultural Research Organization,
POB 6, Bet Dagan 50250, Israel
e-mail: ergraber@volcani.agri.gov.il
Y. Meller Harel : M. Kolton : D. Rav David :
M. Borenshtein : Y. Elad
Institute of Plant Protection, The Volcani Center,
Agricultural Research Organization,
POB 6, Bet Dagan 50250, Israel
M. Kolton
Institute of Plant Sciences and Genetics in Agriculture,
The Robert H. Smith Faculty of Agriculture,
Food and Environment,
P.O. Box 12, Rehovot 76100, Israel
increases in plant growth and productivity, the
rhizosphere of biochar-amended pepper plants had
significantly greater abundances of culturable
microbes belonging to prominent soil-associated
groups. Phylogenetic characterization of unique bac-
terial isolates based on 16S rRNA gene analysis
demonstrated that of the 20 unique identified isolates
from roots and bulk soil from the char-amended
growing mix, 16 were affiliated with previously
described plant growth promoting and/or biocontrol
agents. In tomato, biochar treatments positively
enhanced plant height and leaf size, but had no effect
on flower and fruit yield. The positive impacts of
biochar on plant response were not due to direct or
indirect effects on plant nutrition, as there were no
differences between control and treatments in leaf
nutrient content. Nor did biochar affect the field
capacity of the soilless mixture. A number of organic
compounds belonging to various chemical classes,
including n-alkanoic acids, hydroxy and acetoxy
acids, benzoic acids, diols, triols, and phenols were
identified in organic solvent extracts of the biochar.
We conjecture two related alternatives to explain the
improved plant performance under biochar treatment:
(i) the biochar stimulated shifts in microbial popula-
tions towards beneficial plant growth promoting
rhizobacteria or fungi, due to either chemical or
physical attributes of the biochar; or (ii) low doses
of biochar chemicals, many of which are phytotoxic
or biocidal at high concentrations, stimulated plant
growth at low doses (hormesis).
482
Keywords Soilless media . Biochar . Pepper .
Tomato . Plant growth promotion . PGPR . PGPF . Tar .
Intensive agriculture . Fertigation . Plant productivity .
Hormesis . Biocontrol . Antibiotics . Bacterial isolates
Introduction
There is a world-wide drive to develop affordable
energy from renewable resources in order to reduce
net greenhouse gas emissions and diversify energy
sources. Biomass surpasses many other renewable
energy sources due to its abundance, high energy
value, and versatility (Digman et al. 2009). However,
much first generation biofuel production (i.e., ethanol
from sugar and starch crops, and biodiesel from oil
crops) in temperate climates is almost entirely carbon
positive due to heavy inputs of fossil fuels for
agricultural production (Bruun and Luxhoi 2008; Lal
2005; Searchinger et al. 2008). As a result, there is
continued interest in developing second and third
generation biofuels which will be at least carbon
neutral, if not carbon negative (Mathews 2008).
Pyrolysis of waste biomass is one such method; it
involves the thermal decomposition (exothermic) of
biomass in the absence of oxygen to solid (charcoal,
or biochar), liquid (bio-oil) and gas biofuels. Instead
of burning the produced biochar for energy, biochar is
applied to the soil as a conditioner, where it remains
in an essentially permanent form and leads to net
removal of carbon from the atmosphere (Laird 2008;
Lehmann 2007). As a soil additive along with organic
and inorganic fertilizers, biochar has been reported to
significantly improve soil tilth, nutrient availability to
plants, and plant productivity (Glaser et al. 2002).
Pre-Columbian Amazonian Indians used charcoal to
enhance soil productivity, and it is still found in large
concentrations in fertile Amazon soils abandoned
thousands of years ago. Currently, however, very
little biochar is utilized in modern agriculture, and its
agronomic value in terms of crop response and soil
health benefits has yet to be quantified.
For the most part, the available literature docu-
ments a general improvement in plant response under
biochar amendment (Chan et al. 2007, 2008; Glaser et
al. 2002; Iswaran et al. 1980; Steiner et al. 2007;
Wardle et al. 1998), although there are notable
exceptions (Kishimoto and Sugiura 1985; Van
Zwieten et al. 2010). The wide range of reported
Plant Soil (2010) 337:481–496
plant responses (e.g., −29 to 324% increase in dry
matter) can be attributed to the large variability in
studied systems (Glaser et al. 2002). In particular, it is
worth noting that the effect of biochar amendments is
often examined in poor, depleted soils (Lehmann et
al. 2003; Rondon et al. 2007; Steiner et al. 2008; Van
Zwieten et al. 2010) or in systems with sub-optimal
fertilization regimes (Chan et al. 2008; Hossain et al.
2010; Van Zwieten et al. 2010).
The specific mechanisms underlying the contribution
of biochar to plant response are poorly understood.
Regional conditions including climate, soil chemistry
and soil condition affect biochar agronomic benefits. In
addition, different biomass feedstocks and pyrolysis
conditions create biochars with different physical and
chemical properties (Keiluweit et al. 2010), affecting
plant response (Chan et al. 2007, 2008). Biochar can
improve plant productivity directly as a result of its
nutrient content and release characteristics, as well as
indirectly, via: (i) improved retention of nutrients
(Lehmann et al. 2003; Wardle et al. 1998); (ii)
improvements in soil pH (Rondon et al. 2007); (iii)
increased soil cation exchange capacity (Liang et al.
2006); (iv) improved soil physical properties (Chan et
al. 2008), including an increase in soil water retention
(Laird et al. 2010); and (v) alteration of soil microbial
populations and functions (Pietikainen et al. 2000).
These effects may also act in concert to result in
improved crop performance.
Given the complexity of the soil-plant-water-envi-
ronment system, it is difficult to isolate those factors that
actually play an instrumental role in the ‘charcoal effect’
(Wardle et al. 1998). Our goal in the present study was
to test whether biochar can impact plant growth when
nutritional and soil physical aspects of biochar amend-
ment are neutralized. This was achieved by examining
the impact of a nutrient-poor, wood-derived biochar on
tomato (Lycopersicum esculentum Mill.) and pepper
(Capsicum annuum L.) development in a commercial
coconut fiber:tuff soilless mixture under an optimal
fertigation (fertilization plus irrigation) regime in a
greenhouse. If improved plant growth would be
observed under such optimal conditions, it would
demonstrate that biochar-induced plant growth stimu-
lation goes beyond obvious contributions to plant
nutrition and improved soil physical and chemical
properties. This would then provide an opportunity to
evaluate specific factors potentially responsible for the
‘charcoal effect’.
Plant Soil (2010) 337:481–496
Materials and methods
Biochar and plant growth medium
Biochar prepared from citrus wood in a traditional
charcoal pit (lump charcoal) was obtained, ground into
a powder of less than 0.5 mm particles, and stored in a
sealed metal box until use. Plants were grown in a
commercial soilless mixture composed of a mixture of
coconut fiber and tuff at a 7:3 v:v ratio (the tuff is
unsorted to a size of 8 mm). Biochar at the desired rates
(1, 3 or 5% by weight) was mixed into the commercial
soilless mixture. The influence of biochar at 4 amend-
ment levels (0, 1, 3, 5% w:w) on the water holding
capacity of the soilless mixture at field capacity was
tested gravimetrically as follows. Pre-weighed 0.5 L pots
were filled with a known weight of media to a certain
bulk density, the filled pots were weighed, and the
porous media in each was then thoroughly saturated with
a large excess of water. The pots were left to drain freely,
and their weight was measured every 30 min until it
ceased to change (2 h). Field capacity was calculated by
comparing the weight of the media at 2 h with the dry
weight of the media. There were five replicates (n is
number of replicates) per amendment level.
Biochar characterization
The biochar was characterized chemically and phys-
ically in order to define the material as fully as
possible. Aqueous extracts of biochar (1:10 w:w)
were prepared in double distilled water by stirring at
25°C for 30 min, and by autoclaving (121°C, 1 atm.,
35 min.). The extracts were filtered through a
0.22 μm filter (Milex-HV, Millipore) and stored at
4°C until analyzed. The extracts were characterized
for pH, electrical conductivity (EC), nutrient content
(B, Ca, Cu, Fe, K, Mg, Mn, P, S, Si, Zn, Na) by
inductively coupled plasma (ICP-AES Arcos, EOP
model, Spectro Ltd.), Cl by digital chloridometer
(Labconco), N-NO3 and N-NH4 by injector autoan-
alyzer (Lachat Instruments), and total organic carbon
(TOC) content by total organic carbon analyzer
(Skalar Analytical B.V.). A concentrated acid extract
(0.1 g biochar in 10 mL 70% HNO3, digested at
120°C for 24 h) was also analyzed by ICP for B, Ca,
Cu, Fe, K, Mg, Mn, P, S, Zn, and Na.
Ash content (in duplicate) was determined by
weight loss after heating to 800°C in air for 4 h.
483
Total C, H, N, O, and S were determined in triplicate
by element analyzer (Thermo Flash EA-1112 Ele-
mental Analyzer). X-ray diffraction analysis was
carried out on a Philips XRD diffractometer, and
biochar samples (both uncoated and gold coated)
were observed by scanning electron microscope
(SEM; model JSM-5410, JEOL Ltd). Element iden-
tification on uncoated samples was by Link ISIS X-
Ray Analysis detector (Oxford Instruments). FTIR
absorbance spectra of KBr pellets prepared with 0.6%
wt biochar were recorded between 400 and
4000 cm−1 with one hundred scans averaged with a
resolution of 4 cm−1 (Bruker Tensor 27 FTIR
Spectrometer). The specific surface area (SSA) of
the biochar was determined by N2-BET adsorption at
The Israel Ceramic & Silicate Institute, Technion
City, Haifa, Israel.
Biochar tars were extracted by automated Soxhlet
(VELP) in a dichloromethane:methanol (DCM:
MeOH) 95:5 v:v mixture. Biochar (0.5 g) was boiled
for 1 hr in 40 mL of solvent and rinsed for 1 h in the
condensed solvent vapors. The solvent extract was
then evaporated to dryness and derivatized using
0.5 mL of N,O-Bis(trimethylsilyl)trifluoroacetamide
(BSTFA) and 1 mL acetonitrile for 15 min at 60°C.
Analysis was carried out by gas chromatography/mass
spectrometry (GC/MS; Agilent, model number
6890N/5973N) in scan mode using temperature
programming (oven 170°C, initial hold 5 min, ramp
to 300°C at 5°C/min, final hold 10 min) and a 30 m
long capillary column with a (5%-phenyl)-methylpo-
lysiloxane phase, 0.25 mm inner diameter, and
0.25 μm film thickness. Compound identification
was via the NIST98 library.
Plants and fertigation regime
Plants of tomato cv. 1402 (Hazera Genetics, Ltd.,
Brurim, M.P. Shikmim, Israel) and sweet pepper cv.
Maccabi (Hazera Genetics, Israel) were obtained from
a commercial nursery (Hishtil, Ashkelon, Israel) at
40–50 days after seeding, and transplanted into 1-L
pots containing the potting medium without or with
biochar at 1, 3 or 5% by weight. Plants were
fertigated with drippers 2–3 times per day with
5:3:8 NPK fertilizer (irrigation water prepared to have
N (total), P and K concentrations of 120, 30, and
150 mgL−1, respectively; EC 2.2 dS m−1), allowing
for 25–50% drainage. Plants, trained on bamboo
484
sticks or hooked to polyethylene ropes as training
systems, were maintained at 20–30°C in a pest- and
disease-free greenhouse for up to 3 months.
Experimental design and statistical analysis
A total of six sets of greenhouse experiments were
carried out. Two sets (one for each crop pepper (P)
and tomato (T)) compared 0, 1, and 3% biochar by
weight (experiments 013P [n=9] and 013T [n=7]),
two sets compared 0, 3, and 5% biochar by weight
(experiments 035P [n=6] and 035T [n=6]), and two
sets compared 0 and 5% biochar by weight (experi-
ments 05P [n=10] and 05T [n=10]). Replicate plants
in each experimental set were arranged randomly in
the greenhouse. Data was averaged per leaf level
(node), plant, and replicate. In order to compensate
for initial variability in plant parameters, individual
data at each evaluation date is presented as the percent
change compared with the value at the initial
evaluation of the same plant organ. Calculation of
data at each sampling date was carried out according
to the formula C=100×B/A where A=value at initial
evaluation date, B=value at a given evaluation date,
and C=calculated percentage of change. Data in
percentages was arcsin-transformed before further
analysis. Data was analyzed using ANOVA and
Fisher’s protected LSD test. Standard errors (SE) of
the means were calculated and growth levels were
statistically separated following a one-way analysis of
variance. The standard errors (SE) are marked in the
figures with error bars and stated in the tables and
text. Results marked with different lower case letters
are statistically different at a significance level of P≤
0.05. Statistical analysis was done using R version
2.10.1 software (http://www.r-project.org).
Plant growth parameters
Plant growth parameters were evaluated every 1–
3 weeks or when plants reached a stage for evaluation
(e.g., presence of mature leaves, blossoms, set fruits,
and mature fruits). Plant height was measured from
stem base to top. Leaves at a certain fixed node height
were marked and evaluated. Area of leaf blade
(pepper) and terminal leaflet (tomato) were calculated
from width and length measurements according to
the formula A=W×L×R where W and L refer to
width and length and R is a constant factor equal
Plant Soil (2010) 337:481–496
to 0.78 and 0.85 for tomato and pepper leaves,
respectively. These factors were derived by measur-
ing width, length and area of 25 leaves of each plant
type. The length of the leaf axis was measured in
tomato leaves.
For flower and fruit counting, buds were regarded
as flowers as long as fruit set was not visible. Fruits
were regarded as set fruits until they reached the size
of 12 mm diam. Fruits were harvested when they
reached the color of mature grade according to
common practice. Canopy fresh weight of pepper
plants was evaluated after detaching the fruits. Plant
material was dried in an air circulation 60°C oven for
5–6 days until dry weight was unchanged.
Leaf nutrient sampling and measurements
Leaves (1 each from the top and bottom of each plant)
were sampled 35, 52, and 65 days following planting
in experiment 013P, and 52 and 65 days following
planting in experiment 013T. Leaves were washed
with distilled water, dried for 1 week in a ventilated
oven at 60°C, and stored pending chemical analysis.
The dry tissue was ground to pass a 20-mesh sieve,
and 100-mg samples were wet ashed with H2SO4-
H2O2 and analyzed for Na, K, organic-N, and P.
Ashing in HClO4-HNO3 was used to analyze for Ca,
Mg, and micronutrients. Element concentrations were
determined as follows: NO3-N and P by autoanalyzer;
K by flame photometer (M410, Sherwood Sci. Ltd);
Ca, Mg, Fe, Zn, and Mn by Analyst 800 atomic
absorption spectrophotometer (Perkin Elmer).
Culturable microbe abundances in pepper
experiments
From experimental set 013P, potting medium clinging
to root surfaces (rhizosphere) and located around the
roots (bulk medium) was sampled non-destructively
from 3 replicate plants 76 days after planting, and
from 3 other replicate plants 84 days after planting.
Replicates were not pooled. A 10 g (wet weight)
aliquot of the sampled bulk medium was suspended in
4.5 ml of sterile distilled water by gentle agitation
(25 rpm) for 30 min at 20°C. In parallel, sampled
roots were washed under tap water to remove the bulk
of the attached soil, excess water was thoroughly
blotted off, and 10 g (wet weight) were gently crushed
with a pestle in a clean plastic bag. The root tissue
Plant Soil (2010) 337:481–496
was then transferred to a sterile Erlenmeyer vial
containing 4.5 ml of sterile distilled water and
subjected to gentle agitation as described above.
Ten-fold serial dilutions were prepared from each
sample. Three replicate 100 μL aliquots from each
dilution were plated for fungi and bacteria on agar
media as detailed below.
General bacterial were quantified on nutrient agar
supplemented with 1 ppm benomyl (Dhingra and
Sinclair 1986), Pseudomonas spp. on Kings B
medium (Elad and Baker 1985), filamentous fungi,
yeast and Trichoderma spp. on PDA medium supple-
mented with 50 ppm Rose Bengal (4,5,6,7-tetra-
chloro-2′,4′,5′,7′-tetraiodofluorescein) stain (Elad et
al. 1981), and actinomycetes species on alkaline water
agar (Ho and Ko 1980). Bacillus spp. were quantified
on nutrient agar following boiling for 20 min to kill
vegetative cells but not endospores (Dhingra and
Sinclair 1986). Microbe abundances are presented as
colony forming units (CFU) per g sample. Results
from the two sampling events were similar, therefore,
those from the first event are presented.
Taxonomic characterization of isolated bacteria
Bacterial isolates from biochar-amended and control
potting mixtures showing distinct colony morphologies
were characterized based on 16S rRNA gene sequence
phylogeny. Colonies were resuspended in 100 μL of
sterile distilled water, boiled for 10 min and centrifuged
at 15000 g for 5 min at room temperature. PCR
amplification was performed using 2.5–5 μl of the
supernatant as a template with the general bacterial
primer pair 11F (5′GTTTGATCCTGGCTCAG (Kane
et al. 1993)) and 1392R (5′ACGGGCGGTGTGTRC
(Lane 1991)). PCR reactions (final volume of 50 μL)
contained the following components: 1.5 U Taq DNA
polymerase (DreamTaq; Fermentas), Taq buffer con-
taining a final magnesium concentration of 2.5 mM,
dNTPs (20 nmol each), 12.5 μg bovine serum albumin
and 25 pmol of each primer. The PCR program was
carried out as previously described (Cytryn et al. 2006)
with slight modifications: an initial denaturation step of
95°C for 180 s followed by 30 cycles of denaturation
at 95°C for 30 s, annealing at 55°C for 30 s and
elongation at 72°C for 80 s. Cycling was completed
with a final elongation step at 72°C for 5 min. The
presence and size of PCR amplicons were determined
by agarose gel electrophoresis (1%). The PCR products
485
of colonies that gave a single distinct band were
sequenced using the general bacterial primer 907R (5′
CCGTCAATTCMTTTGAGTTT (Muyzer et al. 1998).
Partial 16S rRNA gene sequences (630 bp) were
characterized using the Ribosomal Database Project
(RDP) on-line sequence analyses package. After
elimination of chimeric sequences using Chimera-
CHECK (Cole et al. 2009), partial 16S rRNA gene
sequences were compared to sequences from the
GenBank and RDP databases using Blast and the
RDP Seqmatch online classifier (Wang et al. 2007)
(www.ncbi.nlm.nih.gov/BLAST/ and www.rdp.cme.
msu.edu respectively). Sequences were deposited into
the Genbank nucleotide sequence database under
accession numbers HM481450-HM481472.
Results
Biochar characterization
The biochar exhibited very weak absorbance in the IR
spectrum, with the major observable bands of the FTIR
spectrum occurring at about 1600 and 1430 cm−1
denoting aromatic ring C=C stretching, and a weaker
band at about 1700 cm−1, indicative of aromatic
carbonyl/carboxyl C=O stretching (Guo and Bustin
1998) (Fig. 1a). Ash content of the biochar was 10.9%.
The only definable mineral phases identified by X-ray
diffraction of the biochar were quartz and calcite, both
at low, non-quantifiable levels. These results corre-
sponded well with SEM-EDX results, which did not
reveal any separate mineral phases. The pore structure
of the precursor wood can clearly be seen in the SEM
images (Fig. 1b). The surface area of the biochar, as
determined by N2-BET, was 46.2 m2 g−1. Despite its
porous nature, the biochar had no impact on the water-
holding capacity of the soilless media at any amend-
ment level (measured field capacity 31.9±4.6, 31.9±
3.1, 30.4±2.2, and 32.4±4.5%, for 0, 1, 3 and 5%
biochar, respectively). Accounting for ash content, the
elemental composition of the biochar was found to be
70.6% C, 0.6% N, 2.3% H, and 15.5% O, giving an O/
C atomic ratio of 0.16, H/C ratio of 0.40, C/N ratio of
130.69, and an H/O ratio of 2.41. These results are
similar to those for wood chars reported in the
literature (Krull et al. 2009).
The chemical characterization of the biochar aqueous
extracts is presented in Table 1. pH of the extracts was
486
Fig. 1 a Biochar FTIR
spectrum; b SEM photomi-
crograph of a typical
biochar particle
nearly neutral, EC was lower than the EC of the
fertigation solution (2.2 mS m−1), and of the essential
macronutrients (NPK), only K existed in substantial
quantities (10%). As expected, because of its higher
temperature, the autoclave extraction was more effec-
tive than the 25°C aqueous extraction for release of
most of the measured elements. By weight, only 50%
of the biochar was digested in the hot concentrated
HNO3; Ca and Fe were present in the greatest
concentrations (1.8 and 1.1%, respectively). Potassium,
Na, and S were the only elements released from the
biochar to the aqueous solutions in relatively large
amounts as compared with their amount in the HNO3
digest, and were likely present in the biochar as readily
soluble salts. As such, the biochar could not serve as a
long-term source of these elements despite their high
concentrations in the aqueous extract.
DCM:MeOH extracts of the biochar contained a
number of identifiable organic compounds belonging
to various chemical classes, including n-alkanoic
Plant Soil (2010) 337:481–496
acids, hydroxy and acetoxy acids, benzoic acids,
diols, triols, and phenols. Identified compounds are
depicted in Table 2.
Greenhouse experiments
Results from experiments 013P, 013T, 035P, and
035T are presented below. Many fewer plant growth
indicators were measured for experimental sets 05P
and 05T, thus, while the results revealed similar
trends to the other 4 sets, they are not reported here.
No deleterious effects of biochar amendment on plant
development were noted in any experiment.
Plant height, number of nodes, and canopy The
influence of biochar amendment (0, 1, and 3%) on
tomato plant height (experiment 013T) over time, is
presented in Fig. 2a, with results normalized to height
at the first measurement (day 35). Normalization
served to account for any unintended differences
Plant Soil (2010) 337:481–496 487

Table 1 Biochar element composition

Element H2O-25°C extract (mg g−1 char) H2O-autoclave extract (mg g−1 char) HNO3 digestion (mg g−1 char)

B 0.001 0.003 0.061

Ca 0.079 0.204 17.729
Cu 0.000 0.000 0.039
Fe 0.000 0.000 10.905
K 2.740 3.690 6.829
Mg 0.029 0.112 2.390
Mn 0.000 0.000 0.145
P 0.008 0.008 0.919
S 0.607 0.691 1.555
Si 0.005 0.044 n.d.
Zn 0.000 0.000 0.505
Na 2.335 3.162 4.785
Cl 3.240 4.467 n.d.
N-NO3 0.0006 – –
N-NH4 0.0002 – –
pH# (units) 7.55 – –
EC# (dS m−1) 1.6 – –
TOC# 79.7 – –

n.d. not determined

#
given for 1:10 w:w aqueous extract

between the treatments at the time of planting.
Tomato plant heights were significantly greater in
the 1 and 3% biochar treatments at all measurement
times as compared with the control (0%), with no
difference between the two levels of biochar amend-
ment (Fig. 2a). Considering both levels of biochar
together, tomato plants were on average 39% taller
than the plants grown in the control treatment at the
final measurement (63 days after planting). Tomato
plants grown in experiment 035T were also signifi-
cantly higher in the biochar treated pots as compared
with the control pots, with the exception of the final
day of measurement, where the height of the 5%
treatment was the same as that of the control
(Fig. 2b). Differences, while significant, were smaller
than in the 013T experimental set (Fig. 2a). This is
presumably because different stages of growth were
followed in the two experiments (note the difference
in time after planting between Fig. 2a, b).
Pepper plant height was not significantly different in
any of the experimental sets (not shown). There were,
however, a significantly greater number of leaf nodes on
the pepper plants in both experiments 035P and 013P.
For example, at 32 days following planting, pepper
plants grown in 0, 3, and 5% biochar (035P) had
12.7±0.4, 14.0±0.3, and 14.8±0.3 nodes, respectively
(average±SE). In experiment 013P at day 64 following
planting, plants grown in 0, 1, and 3% biochar had
14.1±0.1, 15.0±0.3, and 15.0±0.2 nodes, respectively. A
greater number of nodes at a given time implies that the
plants developed more rapidly. Pepper plant canopy dry
weight (035P) was also significantly greater in the
biochar amended treatments (Fig. 3a), while differences
in canopy weight in experiment 013P were not
significant (not shown).
Leaf area and length Leaf area of both pepper and
tomato plants showed significant responses to biochar
treatments at all levels (1, 3, and 5%) as depicted in
Figs. 3 and 4. In Fig. 3b and c, changes in pepper leaf
area normalized to the first measurement are shown
for pepper leaves at the different biochar treatment
levels for leaves at node 12. Leaf area at all biochar
treatment levels (013P and 035P) was significantly
greater than in comparable leaves of the control
plants, with the leaves from all the biochar treated
488 Plant Soil (2010) 337:481–496

Table 2 Organic compounds in dichloromethane:methanol (95:5 v:v) extract of biochar

Alkanoic Acids
O O

OH n OH

3-methyl-butyric acid n-alkanoic acids: 6:0, 9:0, 10:0, 12:0, 14:0, 16:0, 18:0

Hydroxy and AcetoxyAcids

O
O O OH O
OH

HO
O OH OH

3-acetoxy-butyric acid 2-hydroxy-propionic acid 3-hydroxy-butyric acid

Diols and Triols

HO HO OH
HO OH
OH

ethane-1,2-diol propane-1,2-diol 2-methyl-propane-1,3-diol

OH OH

OH

HO OH HO
1,2,3-propantriol 1,2, 4- trihyd ro xy but an e

Benzoic Acids
HO
O

O
HO
benzoic acid 2-m ethyl-be nzo ic aci d

Phenols
OH
OH
HO

HO OH HO

phenol 2-methyl-phenol 1,3-dihydroxybenzene 1,4-dihydroxybenzene

(o-cresol) (recorsinol) (hydroquinone)

Others

octadecane

HO
O
2-phenoxy-ethanol
Plant Soil (2010) 337:481–496 489
Fig. 2 Effect of biochar on

Normalized tomato plant height (%)

400 a
tomato plant height (a), 700 a 013T
a
b 035T b
013T, and (b) 035T. At a a
given date, statistically sig- 600
nificant differences (P≤
0.05) are denoted by differ- 500 300
a b
ent lowercase letter. Error b a
bars represent standard error 400 b
b
300 200
Biochar (%) Biochar (%)
0 a 0
a
200 1 3
b b
3 5
100 100
35 40 45 50 55 60 65 0 5 10 15 20
Time after planting (days)

plants being between 19 to 25% greater. At higher Buds, flowers and fruits Results for pepper plants in
nodes, differences between the biochar rates became 0, 3, and 5% biochar (experiment 035P) for all
more expressed (compare Fig. 3d to c). Relative buds, set fruits, total fruit weight, and single fruit
terminal leaflet area and relative leaf length of tomato weight are tabulated in Table 3. In general, the 3
plants were also significantly greater in biochar and 5% biochar rates resulted in significant increases
treatments, as shown in Fig. 4 for 013T. Similar for all parameters. Results of experiment 013P with
results were also obtained for 035T (not shown). 0, 1, and 3% biochar also demonstrated a significant

Fig. 3 Effect of biochar on 20 Normalized pepper leaf area (%) 240

a 035P b 013P
Pepper canopy dry weight (g)

(a) pepper canopy dry a

weight (035P); (b) pepper 220
19 a
leaf area (013P) at node 12; a 200 Biochar (%)
(c) pepper leaf area (035P)
18 0 a
at node 12; and (d) pepper 180 1 b
leaf area (035P) at node 15. 3
At a given date, statistically 17 160 b
significant differences (P≤ b
140
0.05) are denoted by differ- 16 node 12
ent lowercase letters. Error 120
bars represent standard error 15
1 100
0
0 3 5 65 70 75 80 85
Biochar Content (%) Time after planting (days)
Normalized pepper leaf area (%)

Normalized pepper leaf area (%)

450
c 035P 360 d 035P
a
400 320 a
Biochar (%) a Biochar (%)
350 0 280 0
a a a
3 b 3 b
300 5 b b
240 5 c
b b
250 c
200
200 c
160
node 12 node 15
150
120
100

30 35 40 45 50 55 60 30 40 45 50 55 60
Time after planting (days) Time after planting (days)
490 Plant Soil (2010) 337:481–496
Fig. 4 Effect of biochar in 225

Normalized tomato leaf length (%)

Normalized tomato leaflet area (%)
experiment 013T on (a) to- 300 a 013T b 013T
mato leaflet area, and (b) a 200
tomato leaf length. At a a a
a b
given date, statistically sig- 250 a b
a ab c 175
nificant differences (P≤ ab c
0.05) are denoted by differ- b
200 b
ent lowercase letters. Error ab b 150
bars represent standard error b
150 Biochar (%) Biochar (%) 125
0 0
1 1
100 3 3 100

40 45 50 55 60 65 40 45 50 55 60 65
Time after planting (days)

increase in number of flowers per pepper plant and 15.4±0.3, and 6.8±0.4 g kg−1 DW, respectively, and
number of mature peppers per plant for both biochar leaf-Fe, -Zn, and -Mn concentrations were 157±21,
doses (Table 4). Total pepper fruit yield per plant and 101±19, and 163±45 μg kg−1, respectively. Results
single fruit weight were also significantly greater in of leaf nutrient analyses demonstrate that despite the
the biochar treated plants, with the 3% treatment high C/N ratio of the biochar, there was no N-
significantly outperforming the 1% treatment deficiency in either crop at any biochar level,
(Table 4). Tomato plant bud, flower and fruit yields indicating negligible N immobilization under the
did not exhibit significant effects of biochar (data not prevailing fertigation conditions. In addition, the
shown). results demonstrated that any nutrients provided by
the biochar (Table 1) were negligible in comparison
Leaf nutrient content Concentrations of nutritional to the nutrients supplied equally to all the treatments
elements in tomato and pepper leaves were within and controls in the fertigation solution.
the optimal range for these plants (Adams 2002;
Sonneveld 2002), and there were no significant Abundances and taxonomic characterization of cul-
differences between the various treatments and turable microbes in pepper experiments Abundances
controls at any sampling event for either crop. Mean of culturable root-associated fungi and bacteria
± SE N, P, K, Ca, and Mg concentrations in tomato were measured only in experiment 013P (0, 1, and
leaves were 32.1±3.1, 5.6±1.7, 37.4±7.4, 35.7±0.4, 3% biochar, pepper). Results are given in Table 5.
and 10.3±0.8 g kg−1 DW, respectively, and leaf-Fe, Root-associated yeast and Trichoderma spp., which
-Zn and –Mn concentrations were 149±12, 49±6, were non-measurable in the control treatment, increased
and 109±3 μg kg−1, respectively. In pepper plants, by 3 and 2 log units in the biochar treatments,
mean ± SE pepper leaf N, P, K, Ca and Mg respectively. Significantly greater numbers of general
concentrations were 46.4±2.3, 3.8±0.3, 74.5±13.4, bacteria, Pseudomonas spp. and fungi were also

Table 3 Effect of biochar amendment on bud and fruit formation in pepper, experiment 035P

Biochar (wt %) Buds (number plant−1) Set fruits (number plant−1) Fruit weight

Whole plant yield (g) Single fruit (g)

Time after planting (days)

32 37 44 57 57

0 13.5±0.4 b 15.0±1.1 b 3.0±0.9 b 440.0±21.1 b 83.3±7.2 b

3 17.5±1.0 a 17.5±1.5 a 5.2±1.1 ab 565.0±36.1 b 97.2±4.6 a
5 17.5±1.7 a 17.5±2.1 a 7.2±0.9 a 732.0±58.5 a 104.8±4.2 a
Plant Soil (2010) 337:481–496 491

Table 4 Effect of biochar amendment on bud and fruit formation in pepper, experiment 013P

Biochar (wt %) Buds (number plant−1) Mature fruits (number plant−1) Fruit weight

Whole plant yield (g) Single fruit (g)

Time after planting (days)

54 64 64 64

0 3.6 ±0.8 b 2.0±0.6 b 201.4±25.8 c 99.1±3.0 b

1 6.9 ±0.7 a 3.1 ±0.5 a 359.6±29.2 b 115.1±4.4 ab
3 7.4 ±0.4 a 5.5±0.9 a 662.5±112.0 a 119.7±2.1 a

observed in the bulk potting mixture in the biochar-
amended treatments relative to the bulk potting
mixtures of the control treatment (Table 5). In general,
the biochar enhanced abundances of cultured microbes
were more pronounced on root surfaces than in the
bulk medium (Table 5).
Phylogenetic characterization of unique bacterial
isolates from the biochar-amended growing mixtures
based on partial 16S rRNA gene analysis is shown in
Table 6. Of the 20 unique identified isolates, 16 were
affiliated with previously described plant growth
promoting or biocontrol agents (see references listed
in Table 6).
Discussion
It is seen here that modest additions of a nutrient-poor
biochar added to a commercial coconut fiber:tuff

Table 5 Culturable microbe

abundances from experiment
013P
0.0=below detection level
Microorganism group Biochar (%) Bulk potting mixture Root-associated
General bacteria 0 1.25∙107 ±1.00∙105 b 1.92∙107 ±1.42∙106 b
7 6
1 1.45∙10 ±144∙10 b 3.60∙107 ±2.47∙106 b
3 3.53∙107 ±2.68∙106 a 4.32∙107 ±1.64∙106 a
Bacillus spp. 0 0.0±0.0 a 0.0±0.0 a
1 16.7±16.7 a 216.7±1.48∙102 a
3 33.3±33.3 a 50.0±28.9 a
Filamentous fungi 0 5.02∙104 ±2.70∙103 b 5.33∙103 ±4.40∙102 b
1 1.26∙105 ±8.50∙103 a 1.10 104 ±5.77∙102 a
5 4 4 3
3 1.29∙10 ±1.50∙10 a 1.27∙10 ±1.45∙10 a
Yeasts 0 1.33∙103 ±8.8∙102 a 0.0±0.0 b
1 1.83∙103 ±8.8∙102 a 4.17∙103 ±1.76∙103 a
3 3.83∙103 ±6∙102 a 6.00∙103 ±8.66∙102 a
2 1
Trichoderma spp. 0 1.67∙10 ±1.7∙10 a 0.0±0.0 b
2 2 2 2
1 2.33∙10 ±1.1∙10 a 4.17∙10 ±1.80∙10 a
3 3.33∙102 ±1.2∙102 a 5.00∙102 ±5.0∙101 a
Pseudomonas spp. 0 4.20∙106 ±5.77∙104 b 4.27∙106 ±1.01∙105 a
1 4.73∙106 ±3.66∙105 b 2.10∙106 ±1.73∙105 b
3 1.60∙106 ±1.15∙105 a 1.25∙106 ±1.25∙106 a
7 7 7 6
Actinomycetes spp. 0 4.50∙10 ±1.76∙10 a 2.16∙10 ±1.66∙10 b
1 1.00∙107 ±5.77∙106 a 3.16∙107 ±1.10∙107 b
3 3.33∙107 ±1.36∙107 a 6.5∙107 ±2.80∙106 a
492 Plant Soil (2010) 337:481–496

Table 6 Phylogenetic affiliation of char-amended potting mixture isolates. Closest defined bacterial relative based on partial 16S
rRNA gene sequence (630 bp). Isolates from 013P

Isolation point Name (acc #) Closest defined related species (% identity) Potential environmental role

Root 1PR3 Pseudomonas mendocina (99%) PGP (Kohler et al. 2010); BC

(Duponnois et al. 1999)
1PR15 Brevibacillus limnophilus (99%); PGP (Zhang et al. 2001); BC
Brevibacillus brevis (99%) (Li et al. 2005)
1PR12 Micrococcus luteus (100%); No known effect
1PR4 Corynebacterium sputa (99%) No known effect
A
1PR16 Streptomyces rochei (99%) BC (Ezziyyani et al. 2007)
3PR14 Bacillus licheniformis (100%) PGP (Lim and Kim 2009); BC
(Garcia et al. 2004)
3PR21 Bacillus firmus (99%) PGP (Ghosh et al. 2003); BC
(Chaiharn et al. 2009)
3PR42 Pseudomonas aeruginosa (99%) BC (Dutta et al. 2008)
Soil 3PS6 Micrococcus sp. M3T3B2 (92%) ND
3PS39 Streptomyces thermocarboxydus (97%) BC (Inbar et al. 2005)
1PS8 Mesorhizobium huakuii (98%) PGP (Contesto et al. 2008)
1PS13 Bacillus amyloliquefaciens (100%) PGP (Ryu et al. 2005); BC
(Choudhary and Johri 2009)
3PS17 Streptomyces hygroscopicus (100%)A BC (Prapagdee et al. 2008)
3PS24 Bacillus cereus (100%), Bacillus thuringiensis (100%) PGP (Raddadi et al. 2008); BC
(Liu et al. 2008)
1PS25 Microbacterium paraoxydans (100%); BC (Pereira et al. 2009)
Microbacterium oleivorans (99%)
1PS27 Nocardioides nitrophenolicus (100%) OCD (Yoon et al. 1999)
1PS28 Streptomyces fradiae (98%)A BC (Srinivasan and
Mathivanan 2009)
3PS29 Agrobacterium radiobacter (100%) BC (Moore 1988)
B
Agrobacterium tumefaciens (100%)
1PS36 Bacillus licheniformis (100%) PGP (Lim and Kim 2009); BC
(Garcia et al. 2004)
3PS41 Mycobacterium fortuitum (96%)C ND

Potential environmental role of isolates: BC (biocontrol); PGP (plant growth promotion); OCD (organic compound degradation) as
determined by previously described studies. Isolates with closest relative sequence identities lower than 97% were not determined
(ND).
A
Share same sequence identity with several additional Streptomyces strains
B
Different agrobacteria strains with identical 16S rRNA gene sequences can be either plant pathogens or biocontrol agents
C
Shared 96% sequence identity with several additional Mycobacterium strains

growing mix had a positive impact on plant perfor-
mance even in systems where porous media structure
was optimized and fertilizer and water were supplied
in excess of plant needs. The positive impacts of
biochar on pepper and tomato plant response were not
due to direct or indirect effects on plant nutrition (no
differences between control and treatments in leaf
nutrient contents), nor to improvements in water
holding capacity of the soilless mixture (no differ-
ences between control and treatments in field
capacity).
Coinciding with the improved plant growth reported
herein, pepper and tomato plants grown in biochar
amended treatments of these same experiments were
shown to be less susceptible to two foliar fungal
pathogens (Botrytis cinerea and Leveillula taurica),
and for pepper, to a foliar mite (Polyphagotarsonemus
latus) (Elad et al. 2010). Given that the biochar was
Plant Soil (2010) 337:481–496
soil-applied and a suppressive effect was found for
foliar diseases, it is apparent that the biochar induced
systemic resistance to disease (Elad et al. 2010), as
well as promoted plant growth.
The question arises, to what may be attributed the
concurrence of improved plant growth and potentiation
of plant systemic resistance to disease in the biochar-
amended systems? We can conjecture two related
hypotheses: (i) the biochar caused shifts in microbial
populations towards beneficial plant growth-promoting
rhizobacteria (PGPR) and fungi (PGPF), many species
of which are also known to induce systemic resistance to
plant diseases; or (ii) compounds in the biochar tars
caused chemical facilitation (hormesis), whereby chem-
icals which are phytotoxic or biocidal at high concen-
trations are able to stimulate growth and induce systemic
resistance to disease at low doses.
Hypothesis I Microorganisms in general, and specif-
ically selected strains belonging to the genera Pseu-
domonas, Bacillus, and Trichoderma, are known for
their ability both to improve plant growth and to
potentiate plant systemic resistance against diseases
and pests in many plant species (Harman et al. 2004;
Kloepper et al. 2004; Gravel et al. 2007; Kaewchai et
al. 2009; Koike et al. 2001; Srinath et al. 2003).
Several PGPRs or PGPFs in combination can also
have a synergistic effect on both plant growth
promotion and biocontrol, as shown, for example,
for Bacillus, Pseudomonas and Chryseobacterium
spp. in tomato and pepper plants (Domenech et al.
2006). Such a synergy could have occurred in the
current study, where culturable microbe abundances
of most of the tested groups were found to be
significantly higher in the rhizosphere of biochar-
amended treatments (Table 5). Several of the root-
associated bacterial isolates from the biochar-
amended potting mixtures shared high sequence
identity to Pseudomonas, Mesorhizobium, Microbac-
terium, Brevibacillus, Bacillus and Strepyomyces
strains well known for their ability to act as either/or
both plant growth promoting agents and biocontrol
agents against a variety of plant pathogens (Table 6).
The inert carbon fraction of the biochar itself is not
expected to have an impact on microbial populations and
population diversity (Tong et al. 2007). However, it is
possible that the observed shifts in the microbial
makeup of the growth medium and rhizosphere were
stimulated by the residual organic tars in the biochar
493
(Table 2). Many of the identified compounds are known
to adversely affect microbial growth and survival at high
concentrations. These include, for example, ethylene
glycol and propylene glycol, hydroxy-propionic and
butyric acids, benzoic acid and o-cresol, the quinones
(recorsinol and hydroquinone), and 2-phenoxyethanol.
At low concentrations, however, it is possible that these
tar compounds promoted the growth of indigenous
microbial populations. Specifically, biochar-associated
organic compounds may suppress sensitive components
of the soil microbiota thereby resulting in proliferation
of resistant microbial communities. One possible indi-
cation of this is identification an isolate with 100%
sequence identity to the nitrophenol-degrader Nocar-
dioides nitrophenolicus. Microorganisms which excel at
degrading toxic organic contaminants generally are
more resistant to a variety of toxic organic compounds.
Also, antibiotic and volatile organic compound pro-
ducers are often resistant to a multitude of antibiotics
(Laskaris et al. 2010; Nodwell 2007). Several antibiotic
producers such as the fluorescent pseudomonads (which
include the P. mendocina and P. aeruginosa strains
detected in this study; Table 6) are known to promote
plant growth and induce systemic resistance (Haas and
Défago 2005; Mazurier et al. 2009; Vespermann et al.
2007). Hence, it is possible that the biochar-associated
organic components selected for a more beneficial
microbial community. In addition, the porous structure
of the biochar (Fig. 1b) may have provided physical
refuge, resulting in increased abundances of beneficial
microorganisms (Warnock et al. 2007).
Hypothesis II The second, related hypothesis, sug-
gests that plant growth and potentiation of systemic
disease resistance were stimulated directly by the low
levels of chemicals in the biochar (Table 2). Evidence
is mounting that hormesis, defined as low dose
stimulation/high dose inhibition by chemicals, oper-
ates in a variety of plants as evidenced by numerous
plant growth indicators, certain metabolic processes,
and incidence of plant disease (Calabrese and Blain
2009). Chemicals from many classes, including phe-
nols, carboxylic and fatty acids, aromatic compounds,
hydrocarbons, and others, have been reported to evoke
inverted U-shaped hermetic dose-response curves in
plants (Calabrese and Blain 2009). This effect has also
been documented for various allelochemicals (Liu et al.
2003), the plant hormone ethylene (Pierik et al. 2006),
various herbicides (Velini et al. 2008; Cedergreen et al.
494
2009), and some antibiotics (Migliore et al. 2010).
Moreover, low levels of phytotoxic compounds in the
root zone were reported to simultaneously improve
plant growth and induce systemic resistance against
disease (Prithiviraj et al. 2007).
Clearly, more research into understanding the
impacts of biochar soil amendments on plant growth
promotion is warranted. Until now, most studies have
focused on direct or indirect effects of biochar on plant
nutrition. Herein, we demonstrated a positive impact of
biochar amendment on plant response for two crops
(pepper and tomato) under an optimal fertigation regime
in a well-structured soilless growth medium, where no
role was played by biochar direct or indirect effects on
nutrition, soil physical attributes or water holding
capacity. Therefore, it is evident that alternative mech-
anisms must have made major contributions to the
observed stimulatory effect of the biochar, the ‘charcoal
effect’. Two alternatives include the possibility that the
biochar stimulated the development of beneficial micro-
organisms which promoted plant growth, or that
chemicals in the biochar directly elicited positive plant
responses. Studies to test the isolated microbes and
biochar-borne chemicals for their potential activity in
promoting plant growth and eliciting disease resistance
in these systems are currently underway.
Acknowledgements The research was funded in part by
Autonomous Province of Trento, Call for Proposal Major
Projects 2006, Project ENVIROCHANGE, and in part by The
Chief Scientist of the Ministry of Agriculture and Rural
Development, Israel, Climate Change Research. The authors
acknowledge the help of Mr. Ran Shulhani in establishing
experiments and treating plants. Comments of the reviewers
contributed substantially to the presentation of these results,
and they are gratefully acknowledged.
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