Soil Biology & Biochemistry 63 (2013) 129e141

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Soil Biology & Biochemistry

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

Chickpea genotypes shape the soil microbiome and affect the establishment of the
subsequent durum wheat crop in the semiarid North American Great Plains
Walid Ellouze a, b, c, *, Chantal Hamel b, Vladimir Vujanovic d, Yantai Gan b, Sadok Bouzid c,
Marc St-Arnaud a
a
Institut de recherche en biologie végétale, Université de Montréal and Jardin botanique de Montréal, 4101 rue Sherbrooke est, Montréal, Québec, Canada H1X 2B2
b
Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, P.O. Box 1030, Airport Road, Swift Current, SK, Canada S9H 3X2
c
Département de Sciences Biologiques, Faculté des Sciences de Tunis, Université Tunis El Manar, Campus Universitaire, Tunis 1060, Tunisia
d
Department of Applied Microbiology and Food Science, University of Saskatchewan, Agricultural Bldg., 51 Campus Dr., Saskatoon, SK, Canada S7N 5A8

a r t i c l e i n f o a b s t r a c t

Article history: Accumulating evidence supports the feasibility of creating biotic soil environments that promote root
Received 6 January 2013 health using selected plant genotypes. Five years of field experimentation conducted in the semiarid
Received in revised form grasslands of North America revealed genotypic variation in the influence of chickpea on the composi-
27 March 2013
tion of the soil microbial community and on the establishment of the subsequent crop. A 2-year
Accepted 1 April 2013
experiment documented the effects of four chickpea cultivars on the arable soil microbiome using cul-
Available online 16 April 2013
tural methods, the cloning and sequencing of soil-extracted DNA, and fatty acid methyl ester profiling.
Cultivar CDC Frontier was characterized by low bacterial biomass, whereas Amit and CDC Anna selected
Keywords:
Cicer arietinum L.
similarly structured bacterial communities but contrasting soil fungal communities. Amit and CDC Anna
Triticum turgidum L. var. durum became colonized by arbuscular mycorrhizal (AM) fungi to the same extent, but the arable soil planted
Plant genotype with CDC Anna hosted the highest level of culturable fungal diversity, whereas the soil planted with Amit
Soil microbial diversity hosted the lowest. The highest diversity of culturable fungi and the richness of AM fungal ribotypes (11)
Soil function were also associated with CDC Anna. Amit was preferentially associated with the antagonist species
Arbuscular mycorrhiza Penicillium canescens. Higher durum wheat stand density was found after CDC Anna than after Amit,
Dryland agriculture indicating that microbial diversity is an important feature of productive soils. The influence of chickpea
Soil water
genotype on the arable soil microbiome and on the establishment of the subsequent durum wheat crop
Semiarid prairie
was related to the soil water reserve at depths of 30e120 cm and was eliminated when the chickpea
Plant breeding
crops experienced drought. Genetic variation in the influence of chickpea on the soil microbiome sug-
gests the possibility of selecting genotypes to engineer beneficial soil biotic environments. Inadequate
levels of soil water could limit the success of this strategy, however, in rainfed cropping systems of the
semiarid grasslands of North America.
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction
Soil-borne diseases reduce the efficiency of agriculture, which
leads to seepage of nutrients, negative effects on air and water
quality and significant yield losses. Most soil-borne pathogens are
* Corresponding author. Semiarid Prairie Agricultural Research Centre, Agricul-
ture and Agri-Food Canada, P.O. Box 1030, 1 Airport Rd., Swift Current, SK, Canada
S9H 3X2.
E-mail addresses: oualid.ellouz@agr.gc.ca, w_ellouze@yahoo.fr (W. Ellouze),
hamelc@agr.gc.ca (C. Hamel), vladimir.vujanovic@usask.ca (V. Vujanovic),
yantai.gan@agr.gc.ca (Y. Gan), sadok.bouzid@fst.rnu.tn (S. Bouzid), marc.st-arnaud@
umontreal.ca (M. St-Arnaud).
normal fungal components of the soil microbiota; thus, they can
hardly be controlled with synthetic fungicides. Plant protection
against soil-borne pathogens relies on crop rotation (Reeleder,
2003). However, options for diversifying cropping systems are
often limited, and the build-up of pathogens in soil under short
rotations explains seed and root rot, damping off of seedlings,
stunting, and crop yield decline (Bennett et al., 2012; Haas and
Défago, 2005). Increasing the resilience of agro-ecosystems
through the creation of soil biotic conditions suppressive to soil-
borne diseases could contribute importantly to the dual goals of
improving the sustainability of food production and meeting the
challenge of increasing global food production by 50% by 2050
(Chakraborty and Newton, 2011; Chaparro et al., 2012).

0038-0717/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.soilbio.2013.04.001
130 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141
A large body of evidence shows that susceptible crop plants do
not develop diseases in certain soils even when they are inoculated
with virulent pathogens (Mazzola and Gu, 2000; Schreiner et al.,
2010; Weller et al., 2002). These soils are referred to as being
disease-suppressive. Bacteria (Mendes et al., 2011; Schreiner et al.,
2010; Weller et al., 2002), fungi (Deacon, 1976; Dewan and
Sivasithamparam, 1989; Duffy et al., 1996; Dunlop et al., 1989;
Larkin and Fravel, 1999; Zriba et al., 1999) or both (Larkin et al.,
1996) can be involved in the suppression of various diseases in
disease-suppressive soils around the world (Weller et al., 2002). In
the semiarid grasslands of North America, two fungi explain the soil
suppressiveness of take-all of wheat (Andrade et al., 1994; Zriba
et al., 1999).
The observation that certain plants can select disease-
suppressive soil microbial communities suggests the possibility of
using plants to create soil microbial communities that are condu-
cive to plant health (Chaparro et al., 2012; Ryan et al., 2009). Plants
can induce disease suppressiveness by promoting the build-up of
microorganisms that antagonize pathogenic species. Plant roots
release materials that feed the microbiota and produce various
bioactive compounds (Berg and Smalla, 2009; Dakora and Phillips,
2002; Ellouze et al., 2011; Fries et al., 1997; Gough and Cullimore,
2011; Tellenbach and Sieber, 2012). Seeding wheat in orchards
has been shown to mitigate apple replant disease, and interestingly,
some wheat genotypes have been shown to be better than others in
mitigating the disease (Mazzola and Gu, 2000). The different in-
fluences of different genotypes of a plant species on the soil
microbiota suggest the possibility of selecting cultivars that pro-
mote root health and efficient use of soil resources.
Genetic variation within a plant species is a prerequisite for the
selection of genotypes. This study tested the influence of genotypic
variation of chickpea on the soil microbiome. To test this hypoth-
esis, an experiment was conducted to examine the influence of
chickpea genotypes on the structure of the soil microbial commu-
nity, as revealed by fatty acid methyl ester (FAME) analysis and by
the abundance of culturable phytopathogenic, mycopathogenic,
antagonistic, and dark endophytic fungal species. The diversity of
arbuscular mycorrhizal (AM) fungi was also examined. The sup-
pression of plant pathogens in disease-suppressive soils is thought
to occur through antagonism during both the saprotrophic and
parasitic growth phases of the pathogens (Weller et al., 2002).
Hence, the selection of chickpea genotypes that promote the pro-
liferation of antagonists in the topsoil layer could improve the
performance of the entire cropping system. We tested this hy-
pothesis in a second field experiment. We tested that the soil legacy
of chickpea genotypes influences the growth of a subsequent crop
of durum wheat. A third objective of the study was to improve
understanding of the selective influence of major chickpea cultivars
on the fungi that naturally occur in cultivated soils of the semiarid
grasslands of North America, which is an important zone of
chickpea production.
2. Materials and methods
2.1. Chickpea genotype impact on the soil microbiome
2.1.1. Experimental design and site description
A field experiment with four genotype treatments and four
repetitions was arranged in a randomized complete block design.
The following chickpea cultivars with contrasting phenotypes and
lineages were used: (1) CDC Xena, a large-seeded kabuli type with
unifoliate leaves; (2) CDC Frontier, a large-seeded kabuli with fern
leaves; (3) Amit, a small-seeded kabuli with fern leaves; and (4)
CDC Anna, a desi type. Kabuli and desi are the main classes of
chickpea seed quality. Kabuli chickpea, also known as garbanzo
bean, produces large cream-colored seeds. Desi chickpea produces
smaller, darker-colored seeds with a thick seed coat. Amit is a
cultivar introduced from Europe. The other three cultivars were
bred at the Crop Development Centre of the University of Sas-
katchewan in Canada. These chickpea genotypes were grown in
2005 and 2006 in 2 m
10 m plots in adjacent sites at the South
Farm of the Semiarid Prairie Agricultural Research Centre, in Swift
Current SK (50 150 N, 107 440 W), both of which were planted with
wheat the year before chickpea was grown. The soil at the experi-
mental site is a Brown Chernozem (an Aridic Haploboroll in the US
soil classification system) with a silt loam texture, an organic car-
bon content of 20 g kg1, and a pH (CaCI2) of 6.8. The daily
maximum temperatures and precipitation during the growing
season (MayeSeptember) were obtained from a meteorological
station located within 500 m of the research plots.
Management practices recognized as best were applied. Seeding
rates were targeting a plant density of 40 plants m2. Seeds were
treated with carbathiin, thiabendazole, and metalaxyl, applied as
CrownÒ (Chemtura AgrosolutionsÔ, Lawrenceville, GA, USA) and
ApronÒ XL (Syngenta), as per the manufacturer recommendations.
Seeds were placed 4 cm deep using a hoe press drill equipped with
C shanks, side band openers, fertilizer boxes, a granular Rhizobium
inoculant box, and a seed splitter. The row spacing was 25.4 cm. The
plots received a blanket application of triple superphosphate (0e
45e0) at a rate of 20 kg P ha1. The plots also received 5.5 kg ha1 of
granular inoculant Nitragin GCÒ (LiphaTech Inc., Milwaukee, WI)
containing a minimum of 100 million viable cells of Mesorhizobium
ciceri per gram. Nitrogen and potassium fertilizers were not
required on these crops and soils and were not applied. Weed
control was achieved using a previous fall broadcast application of
ethalfluralin, a pre-seeding burn-off treatment with glyphosate and
in-crop application of sethoxydim at mid-seedling stage, all as
recommended by the manufacturers. Ascochyta blight was noticed
on the chickpea crops at the mid- to late-seedling stage and was
controlled by four foliar applications of pyraclostrobin (HeadlineÔ,
Bayer Crop Science, USA) at 0.4e0.6 L product ha1, alternated with
applications of chlorothalonil (BravoÔ, Syngenta Crop Protection
Canada, Inc.) at a rate of 3e4 L product ha1, between 5 June and 3
August in 2005 and between 29 June and 3 August in 2006.
2.1.2. Soil and root sampling and plant productivity determination
Roots and topsoil samples were collected from the plant rows at
physiological maturity, using a hand corer (2.5 cm diameter
7.5 cm
length). Four cores were taken and combined to produce one sample
per plot. Roots were freed from the soil, which was well mixed and
homogenized by sieving through 2 mm. The soil samples were
divided into four parts. One part of the soil was used for the deter-
mination of soil moisture. The second part was stored at 12  C for
fatty acid methyl ester (FAME) analysis and DNA extraction, as
described below. The third part was stored at 4  C for fungal culture
isolation. The fourth part of the soil was air dried and used for the
measurement of soil pH in water (Hendershot et al., 1993).
The roots in the soil samples were thoroughly washed on a 2-
mm sieve after sampling to minimize fine root losses. The
chickpea roots were then cut into 1-cm fragments and mixed, and
two 1-g subsamples from each plot were placed in individual
plastic cassettes. The roots were cleared and stained using a solu-
tion of ink and vinegar, as described by Vierheilig et al. (1998). The
percentage of chickpea root colonization by AM fungi and other
fungal endophytes (Abdellatif et al., 2009) was determined using
the gridline intersect method (Giovannetti and Mosse, 1980), and
the values were averaged over technical replicates.
At maturity, two 1-m2 samples were harvested from each plot
by hand for determination of total plant aboveground biomass and
seed biomass, after drying to a constant weight. The maturity date
 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141
was recorded. After each chickpea crop in the fall and before durum
wheat was seeded in the spring, one soil core, 30 mm in diameter
and 120 cm deep, was taken to measure the content and distribu-
tion of water in the soil profile of each plot. The cores were sepa-
rated into 0e15, 15e30, 30e60, and 60e120 cm sections in the
laboratory. Densimetric soil moisture was determined by drying
overnight at 105  C, and soil moisture was expressed in volumetric
units using a soil bulk density of 1.1.
A combination of cultural and molecular methods was used to
depict soil microbial diversity. Fatty acid methyl ester (FAME)
markers were quantified to provide a reproducible evaluation of
soil microbial biomass, in addition to a coarse but wholesome
qualitative measure of the active soil microbial biomass (Frostegård
et al., 2011). A cultural approach was used to obtain fine-scale
taxonomic information about the soil fungi associated with the
chickpea genotype. Soil-borne pathogens are typically culturable.
The possibility of verifying sequence-based fungal identification by
microscopic examination and the availability of isolates for further
experimentation are among the advantages of using cultural
methods. Not all fungi can be grown in culture, however. The study
of biotrophic AM fungi, a group of fungi with an important role in
plant health, requires the use of culture-free methods. The 18S
rRNA gene of AM fungi was targeted because the phylogenetic re-
lationships of this group are well documented for this gene.
2.1.3. Fatty acid methyl ester (FAME) analysis
The abundance of active soil microbial biomass in soil samples
was evaluated by FAME analysis, using the method described in
Hamel et al. (2006). Four grams of soil from each plot (equivalent
dry weight) were extracted in a 9.5-mL mixture of dichloro-
methane (DCM): methanol (MeOH): citrate buffer (1:2:0.8 v/v).
Lipid-class separation was conducted in silica gel columns. The
fatty acids in the phospholipid (PLFA) and neutral lipid (NLFA)
fractions of the soil lipid extracts were transmethylated, and
quantification of FAMEs was performed using gas chromatography.
Individual fatty acids have been used as signatures for various
groups of soil microorganisms (Frostegård and Bååth, 1996; Hamel
et al., 2006; Ruess and Chamberlain, 2010). Measures of the PLFA
18:2u6c and 18:1c were used as indicators of fungal biomass (Ruess
and Chamberlain, 2010), and measures of the PLFA and NLFA
16:1u5 were used as indicators of AM fungi (Balser et al., 2005;
Frostegård et al., 2011). PLFA 3OH-12:0, 2OH-14:0 and 2OH-16:0
were the markers used for Gram-negative bacteria (Cavigelli et al.,
1995), PLFA i-15 meth-17:0þ17:0, a-12 meth-15:0, and i-13 meth-
15:0 were the markers for Gram-positive bacteria (Ruess and
Chamberlain, 2010), and PLFA 15:0 was used as a general bacte-

rial marker (Rezanka and Sigler, 2009; Kharlamenko et al., 1995).
2.1.4. Isolation of culturable fungi
Soil fungal populations were described using dilution plating.
One milliliter from a 104 dilution was spread on potato dextrose
agar (PDA) culture media supplemented with neomycin sulfate
(12 mg L1) and streptomycin sulfate (100 mg L1) (Vujanovic et al.,
2002). Five plates per plot were prepared. The plates were incu-
bated at room temperature in the dark and observed after 3 days.
The three plates with the largest biodiversity were selected. All the
fungal colony-forming units (CFU) developing in these plates were
counted and transferred onto new Petri plates. Fungal cultures
were stored in sterile distilled water in 2-mL containers for further
reference.
2.1.5. DNA sequencing and ITS sequence analysis of culturable
fungal diversity
The fungal isolates were grown in Petri plates containing potato
dextrose agar (PDA) medium, supplemented with neomycin sulfate
131
(12 mg L1) and streptomycin sulfate (100 mg L1) (Vujanovic et al.,
2002). Cultures were classified into 140 morphotypes (St-Germain
and Summerbell, 1996) and identified based on the internal tran-
scribed spacer (ITS) region of the rRNA genes. Fungal DNA extrac-
tion was carried out using an UltraClean microbial DNA isolation kit
(MoBio Laboratories), following the manufacturer’s instructions.
The ITS region of each fungus was amplified by polymerase chain
reaction (PCR) in 25-mL reaction volumes, each containing 11 mL of
sterile distilled H2O, 12.5 mL of Taq PCR Master Mix Kit (Qiagen
Laboratories), 0.25 mL (50 mM stock) of each fungal specific primer
ITS1F (Gardes and Bruns, 1993) and ITS4 (White et al., 1990), and
1 mL of extracted genomic DNA. The amplifications were performed
in an Eppendorf’s Mastercycler ePS gradient thermocycler under
the following conditions: 94  C for 3 min; 35
(94  C, 1 min; 55  C,
1 min; 72  C, 1 min); 72  C, 7 min. Reactions were performed with
negative controls containing no DNA. The resulting PCR products
were electrophoresed in 1% (w/v) agarose gels, stained with
ethidium bromide and visualized under UV light.
Sequencing reactions were performed in a commercial labora-
tory (Genome Quebec Innovation Centre). ITS sequences were
analyzed with the Basic Local Alignment Search Tool (Altschul et al.,
1990) through GenBank (http://www.ncbi.nlm.nih.gov/). Se-
quences were submitted to GenBank under the accession numbers
JF311906eJF311973.
2.1.6. DNA sequencing and SSU sequence analysis of AM fungal
diversity
DNA extraction from 0.3-g soil subsamples was carried out using
an UltraClean soil DNA isolation kit (MoBio Laboratories), following
the manufacturer’s instructions.
A nested-PCR approach was employed to amplify AMF DNA. The
first PCR round, using the primers NS1 (White et al., 1990) and NS41
(Simon et al., 1992), amplified a 1.5-kb fragment of the 18S rRNA
gene. PCR was conducted with an Eppendorf’s Mastercycler ePS
gradient thermocycler in a 25-mL volume consisting of 2.5 mL of
10
PCR buffer, 0.5 mL of 25-mM MgCl2, 0.25 mL of 2.5-units-mL1
Taq DNA polymerase, 0.5 mL of 2.5-mM dNTP, 2.5 mL of 5-mM NS1,
2.5 mL of 5-mM NS41, 0.5 mL of Tween 20 (1%), 1 mL of DMSO, 0.125 mL
of BSA (New England Biolabs), 13.625 mL of dd H2O and 1 mL of
extracted DNA (diluted 1:100). The PCR conditions were as follows:
95  C for 3 min, 35
(94  C for 1 min; 50  C for 1 min, and 72  C for
1 min); and 72  C for 10 min. The PCR products were analyzed by
agarose gel electrophoresis (1.0% w/v agarose), stained with
ethidium bromide, and visualized using a Gel Imaging System
(GelDoc, Bio-Rad Laboratories).
The product of the first PCR round with a visible band was diluted
1:10 and used as template in a subsequent nested PCR round. The
primers for this second stage were AML1 and AML2 (Lee et al., 2008).
PCR was conducted in a 50-mL volume in the following reaction
system: 5 mL 10
PCR buffer, 0.25 mL 2.5 units mL1 Taq DNA poly-
merase, 1 mL 2.5 mM dNTP, 5 mL AML1 (5 mM), 5 mL AML2 (5 mM),
32.75 mL dd H2O and 1 mL product of the first PCR. The conditions for
the second PCR round were: 95  C for 15 min; 30
(94  C for 30 s;
58  C for 40 s, and 72  C for 55 s), and 72  C for 10 min. The PCR
products were analyzed by agarose gel electrophoresis (1.0% w/v
agarose), stained with ethidium bromide, and visualized using a Gel
Imaging System (GelDoc, Bio-Rad Laboratories).
Equal amounts of the PCR products from 2005 and 2006 samples
were combined to obtain a pool of eight amplicon libraries for each
experimental treatment and used to evaluate by cloning the di-
versity of AM fungi, which are non-culturable. Each of the four
pooled DNA samples were then cloned using One ShotÒ TOP10
Chemically Competent Escherichia coli and the TOPO TA cloning kit
(Invitrogen), according to the manufacturer’s instructions. Approx-
imately 50 positive clones were isolated from each of the cloning
132 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141
reactions, sequenced, and compared to reference sequences in
Genbank (http://www.ncbi.nlm.nih.gov/). Plasmid DNA extraction
and sequencing reactions were performed in a commercial labora-
tory (Genome Quebec Innovation Centre). SSU rRNA sequences were
submitted to GenBank under the accession numbers JF340036e
JF340050.
2.1.7. Statistical analysis
The diversity of culturable fungi was assessed using diversity
indices. Simpson’s index of diversity (1  D) (Pielou, 1969) (Equa-
tion (1)) and Shannon’s diversity index (H0 ) (Margelef, 1958)
(Equation (2)) were calculated as shown in the following equations:
Ps
ni ðni  1Þ
i¼1
1D ¼ 1
NðN  1Þ
X
s
ni ni
H0 ¼  ln
i¼1
N N
where ni is the number of CFU of species i, S is the number of species
and N is the total number of individuals in the community. A higher
index reflects higher diversity of the fungal community associated
with the different field-grown chickpea genotypes.
Five functional groups of culturable fungi (potential antagonists,
16 species; dark endophytes, 12 species; mycopathogens, 15 spe-
cies; phytopathogens, 14 species; and saprotrophs, 11 species) were
created, and the sizes of these groups were calculated for each plot
by adding the CFU counts, as suggested by Vujanovic et al. (2007).
The significance of the effect of chickpea genotype on the abun-
dance of individual microbial FAME markers of culturable fungal
species richness and diversity and on the size of functional groups
of culturable fungi based on CFU was assessed by analysis of vari-
ance (ANOVA). The mixed model with blocking as random effect
was used in JMP v.6 (SAS Institute, Cary, NC, USA) to analyze the
data. A P-value of 0.05 was used as the threshold of acceptance of
the significance of effects. The significance of the differences be-
tween treatment means was assessed using the LSMeans Student’s
t option in JMP v.6 when significant treatment effects were found. A
cosine transformation was applied to the values of PLFA i-15
methyl-17:0þ17:0 to meet the requirement of normality of the test
before analysis. Potential antagonist and mycopathogen data were
normalized by rank transformation.
The relationships between the plate counts of the fungi
belonging to the different functional groups were explored using
Spearman correlation analysis of non-transformed data in JMP 6.
The effects of genotype on the structure of the soil microbial
community, as expressed by the FAME profile of soil samples, were
tested using the blocked multi-response permutation procedure
(MRPP) with pairwise comparisons, based on the squared
Euclidean distance (McCune and Mefford, 2011) in PC-ORD v.6
(MjM Software, Gleneden Beach, USA). The influence of chickpea
genotype on the abundance of each FAME marker was analyzed by
ANOVA. The FAME data were analyzed by year, with blocking as
random effect, using JMP v.6. The absence of genotype effect in the
dry year of 2006 prompted the examination of the distribution of
water in the soil profile. Redundancy analysis (RDA) and canonical
correspondence analysis (CCA) (McCune and Mefford, 2011) were
conducted in PC-ORD v.6 to test the relationship between the
abundance of the FAME markers under chickpea influence and the
water content in the soil layers at depths of 0e15, 15e30, 30e60,
and 60e120 cm from the soil surface. The significance of the effects
of blocks on the distribution of water in the soil profile in 2005 was
tested by MANOVA with repeated measures in soil depths, using
JMP v.6.
A blocked MRPP based on Euclidean distance (McCune and
Mefford, 2011) was used to test the significance of chickpea geno-
type effects on culturable fungi. PCA was used to visualize the re-
lationships among FAME markers and genotypes in PC-ORD v.6.
The rare species, i.e., those associated with less than two chickpea
genotypes, were not included in these analyses, and the fungal
species abundance data were Hellinger-transformed before being
subjected to PCA analysis (Legendre and Gallagher, 2001; Legendre
and Legendre, 1998). A randomization test was conducted with the
PCA to assess the significance of axes. The non-transformed fungal
species abundance data were also subjected to CCA in PC-ORD v.6 to
test the significance of the relationship between the species data
and the distribution of water in the soil profile.
The AM fungal sequence profile data were subjected to cluster
(1) analysis using Ward’s method and Euclidean distances in PC-ORD
v.6 (McCune and Mefford, 2011).
(2) 2.2. Chickpea genotype effect on the establishment of a following
durum wheat crop
2.2.1. Experimental design and site description
The influence of chickpea’s soil legacy on a subsequent crop was
tested by seeding durum wheat in field plots planted with chickpea
during the previous growing season. Four chickpea genotypes,
Amit, CDC Frontier, CDC Anna, and CDC Luna, were grown in 2008,
2009, and 2010 and re-cropped with durum wheat AC Avonlea in
2009, 2010, and 2011. The experiment was arranged in a random-
ized complete block design with four blocks. Plants were grown in
2 m
8 m plots at the South Farm of the Semiarid Prairie Agri-
cultural Research Centre in Swift Current SK. The soil was an Orthic
Brown Chernozem with a pH of 6.5 and a texture ranging from loam
to silt loam. The soil nutrient content is shown in Table 1 of the
Supplementary material. The chickpea crops were grown under
contrasting climatic conditions. The monthly means of the daily
maximum temperatures and monthly precipitation during the
period of the experiment (2008 to summer 2011) were obtained
from a meteorological station located within 500 m of the research
plots.
2.2.2. Data collection
A wheat plant count was performed by counting the number of
plants on two meters of rows, i.e., one meter in the 3rd row from
the right side of each plot, close to the front, and one meter in the
3rd row from the left side of each plot, close to the back. Plant
counts were performed at the 4-leaf stage of wheat.
Chickpea maturity dates were recorded each year. After each
chickpea crop in fall and before seeding durum wheat each spring,
one core (30 mm in diameter) was pushed into the soil of each plot
to a depth of 120 cm to measure the content and distribution of
water in the soil profile. Cores were separated into 0e30, 30e60,
60e90, and 90e120 cm sections in the laboratory. The soil moisture
contents and bulk densities were determined by weighing after
drying overnight at 105  C, and the soil moisture was expressed in
volumetric units.
2.2.3. Seeding and plot management
Management practices considered the best were applied to the
chickpea crops, as described above. The 2010 growing season was
unusually humid, and the desiccant diquat (formulated as
RegloneÒ) was applied on 23 September, according to the manu-
facturer’s recommendations, to terminate chickpea growth. Durum
wheat was seeded at a rate of 600 seeds m2, in rows 10 cm apart,
using a modified noble hoe press drill equipped with C shanks, side
band openers and fertilizer boxes. Seeds were treated with car-
boxin and thiram formulated as VitafloÒ 280, according to the
 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141 133

manufacturer’s recommendations. All plots received 43 kg ha1 of
diammonium phosphate (11e51e0) with the seeds, to supply
22 kg P ha1 and 5 kg N ha1. Potassium fertilizer was not required
in this soil and was not applied. Weed control was achieved using a
pre-seeding burn-off treatment with glyphosate and in-crop
application of trakoxydim and bromoxynil on 3 June in 2009 and
2010 and on 6 June in 2011. MCPA was used in the herbicide mix in
2009 and 2010, 2,4-D was used in 2011, and clodinafop-propargyl
was used in 2010.
respectively. The temperature was, on average,1.9  C above normal in
2006, and precipitation amounted to 90.5% of the precipitation
normally received (the 30-year average) at this location, according to
Environment Canada (http://www.climat.meteo.gc.ca/climateData/
canada_f.html). The 2005 growing season was approximately
normal.
3.2. Whole soil microbial diversity analysis based on fatty acid
methyl esters (FAME)

2.2.4. Statistical analysis
The significance of the effect of year and chickpea genotype on
the establishment of a subsequent crop, as revealed by durum
wheat plant density, were assessed by ANOVA in JMP v.6 (SAS
Institute, Cary, NC, USA) using a mixed model with genotype as the
fixed effect and block nested in year and year as random effects. The
significance of the effects of chickpea genotype and year on the
distribution of water at depths of 0e30, 30e60, 60e90, and 90e
120 cm in the soil profile were tested by MANOVA with repeated
measures (in depth), with block nested in year. The significance of
the differences between the means was assessed using the
LSMeans Student’s t option in JMP v.6. Regression analysis was
conducted in JMP v.6 to examine the relationship between water
content at different soil depths under the chickpea crops and the
density of durum wheat plant stands.
3. Results
3.1. Weather and chickpea plant productivity
Chickpea varieties produced similar aboveground straw and seed
biomass, and these biomasses were similarly influenced by the
growth conditions prevailing in 2005 and 2006 (Supplementary
material Table 2). The growing season was dry and warm in 2006
(Supplementary material Fig. 1), limiting plant productivity
(Supplementary material Table 2) and hastening the maturity of the
chickpea plants. CDC Anna, CDC Frontier, Amit, and CDC Xena
matured 15, 19, 23, and 26 days earlier in 2006 than in 2005,
Five PLFA markers were influenced by genotype in 2005. These
were two iso methyl-branched indicators of Gram-positive bacteria
(Ruess and Chamberlain, 2010), one 3-hydroxy-substituted indica-
tor of Gram-negative bacteria (Cavigelli et al., 1995), one odd-

numbered straight-chain bacterial indicator (Rezanka and Sigler,
2009; Kharlamenko et al., 1995), and the indicator of AM fungi
16:1u5 (Ruess and Chamberlain, 2010) (Table 1). All indicators of
AM fungal abundance, i.e., PLFA and neutral lipid fatty acid 16:1u5,
and the percentage of AM root colonization, concurred, suggesting
that Amit and CDC Anna stimulate AM fungal growth. The fungal
markers 18:1c and 18:2 were not influenced by genotype, indicating
that taken as a whole, the soil fungal biomass was similar in the
arable soil of all genotypes. The abundance of FAME markers was
unaffected by chickpea genotype in 2006, the dry growing season.
A blocked multi-response permutation procedure revealed the
effect of chickpea genotype (P ¼ 0.0272) on the structure of the soil
bacterial community, as depicted by the profile of FAME markers, in
2005. The microbial community in the arable soil planted with
Amit was different from that associated with CDC Frontier and CDC
Xena, and the community associated with CDC Xena was different
from that associated with CDC Anna, according to pairwise com-
parisons. The arable soil planted with Amit and to a lesser extent
the soil planted with CDC Anna, contained high amounts of the
different FAME markers (Table 1), indicating that these genotypes
created soil environments conducive to bacterial proliferation. In
contrast, arable soil planted with CDC Frontier generally contained
low amounts of the FAME markers, indicating the presence of low
bacterial biomass under this genotype.

Table 1
Abundance of the fatty acid methyl ester (FAME) microbial markers in the top 0e7.5 cm soil layer in the 2005 chickpea crops, and AMF root colonization levels, soil moisture
content, and soil pH in the 2005 and 2006 chickpea crops at physiological maturity. The significance of the block effect on PLFA is also indicated.

Growing season Pa Variety Block

b
Amit CDC Frontier CDC Xena CDC Anna
PLFA (mg gL1 soil)
3OH-12:0 2005 0.431 1.56 1.75 1.53 2.24 þ
a-12 meth-15:0 2005 0.062 3.86 3.19 3.48 4.00 **
i-13 meth-15:0 2005 0.032* 19.8 a 14.1 b 17.9 ab 18.7 ab *
15:0 2005 0.011* 21.7 a 17.8 b 21.3 a 21.7 a *
2OH-14:0 2005 0.020* 57.8 a 48.4 b 52.3 b 57.4 a *
3OH-14:0 2005 0.130 0.548 0.409 0.330 0.309
16:1u5 2005 0.0152* 14.7 a 12.1 c 13.6 b 14.1 ab *
i-15 meth-17:0þ17:0 2005 0.008** 6.1 a 5.0 b 5.5 ab 6.0 a
2OH-16:0 2005 0.507 1.97 1.75 1.99 1.86
18:2 2005 0.155 14.3 12.7 13.7 14.0 *
18:1c 2005 0.285 20.8 18.0 20.3 20.1 *
NLFA (mg gL1 soil)
16:1u5 2005 0.403 0.752 0.497 0.560 0.806
Other variables
AMF root colonization (%) 2005 <0.000*** 78.3 a 54.8 b 59.3 b 87.3 a
2006 0.874ns 51.8 a 60.5 a 56 a 62 a
Soil moisture (%) 2005 0.008** 10.5 b 15.5 a 10.6 b 11.5 b
2006 0.858ns 3.7 a 4.0 a 3.7 a 3.8 a
Soil pH 2005 0.594ns 5.8 a 5.8 a 5.6 a 5.8 a
2006 0.019* 5.8 ab 5.6 b 5.9 a 5.6 b
a
ns not significant; þsignificant at the 10% level; *significant at the 5% level; **significant at the 1% level; ***significant at the 0.1% level.
b
Least square means are not significantly different according to ANOVA-protected LSMeans Student’s t tests when followed by the same letter on the same row (a ¼ 0.05,
n ¼ 4).
134 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141
Canonical correspondence analysis revealed (P ¼ 0.0130) a
relationship between the bacterial community structure in the top
7.5 cm of the soil planted with chickpea and the spring and fall
levels of soil water (Fig. 1), which were not evenly distributed in the
soil profile (Supplementary material Table 3). The analysis also
revealed the importance of the water content in the uppermost soil
layer (0e30 cm), where the bacterial community originated (the 0e
7.5 cm topsoil layer) and the importance of water located at 30e60
and 60e120 cm depths (Fig. 1), suggesting that the relationship
between the bacteria living in the topsoil layer and soil water re-
serves is mediated by the chickpea plants bridging these soil zones.
Some bacterial markers were positively correlated with soil mois-
ture content, whereas other bacterial markers were negatively
correlated. The relationship between FAME markers in the top 0e
7.5 cm of the soil and the amounts of soil water stored at different
soil depths shown by CCA was not detected by RDA, indicating that
this relationship was nonlinear, as expected along long-range
environmental gradients (Legendre and Legendre, 1998).
The influence of the water content in the top layer of the soil in
spring on the soil microbiome was inversely related to that of the
influence of the soil water in the 0e15, 30e60, and 60e120 cm
layers in fall (Fig. 1).
3.3. Diversity of culturable fungal communities in topsoil planted
with different chickpea genotypes
The 1360 colony-forming units (CFUs) isolated from the arable
soil planted with different chickpea genotypes in 2005 were
separated based on rRNA gene ITS sequence analysis into 68
operational taxonomic units (OTUs) representing different se-
quences (Fig. 2). Most of the fungal taxa were Ascomycetes (92.6%);
Axis 2
i-13meth-15:0
3 was associated with the variety Amit (Table 2). The taxa known for
1 potentially antagonistic fungi was negatively correlated with the
3 1
3 0-15Fall
60-120Fall
3 2
15:0 Axis 1
2 1
30-60Fall 1 blocked MRPP (P ¼ 0.0139). The desi genotype CDC Anna was
2 2OH-14:0
0-15Spring4 2 ogens, specifically Bionectria ochroleuca and Trichoderma sp. (Fig. 3).
16:1 5 4
i-15meth-17:0+17:0
4 planted with CDC Xena. The highest proportion of potentially
4 potentially antagonistic Penicillium taxa (Fig. 3).
Fig. 1. CCA biplot of the analysis of the relationship (P ¼ 0.0130) between the abun-
dance of selected fatty acid (FAME) microbial markers and the amount of water
distributed in the soil profile, showing the differential distribution among blocks
(numbers) of soil water and related FAME markers in 2005. The R2 cut-off threshold,
which was set at 0.2, prevented plotting soil layers with trivial influence. 0e15Spring:
water content of the top 0e15 cm soil layer in spring 2005; 0e15Fall: water content of
the top 0e15 cm soil layer in fall 2005; 30e60Fall: water content of the top 30e60 cm
soil layer in fall 2005; 60e120Fall: water content of the top 60e120 cm soil layer in fall
2005. Axes 1 and 2 explain 61.9% of the variance (N ¼ 40).
others were Zygomycota (5.9%) and Basidiomycota (1.5%). Hypo-
creales was represented by 32 species, Eurotiales by 16 species,
Pleosporales by five species, and Onygenales and Sordariales were
each represented by three species. The order Microascales was
represented by two species, whereas the order Capnodiales and the
undefined order “Incertae sedis” of the family Myxotrichaceae were
each represented by one species.
The antagonistic antibiotic producers Penicillium aurantiogri-
seum, Penicillium canescens, Penicillium griseofulvum, Penicillium
janthinellum, Penicillium spp., Penicillium roseopurpureum, Penicil-
lium tricolor, Penicillium virgatum, and Penicillium ochrochloron were
often abundant (Fig. 2). The dominance of the Penicillium species is at
least partially explained by the fact that cultural methods best
represent heavy-sporulating and fast-growing species. Mycopar-
asitic species constituted the second most abundant group. They
were Bionectria ochroleuca/anamorph Clonostachys rosea, Bionectria,
Clonostachys phyllophila, Hypocrea koningii, Hypocrea lixii/anamorph
Trichoderma harzianum, Hypocrea pilulifera, Trichoderma rossicum,
and Trichoderma sp. The potential fungal pathogens encountered
were Arthrinium, Fusarium acuminatum, Fusarium equiseti, Fusarium
oxysporum, Fusarium redolens, Fusarium solani (teleomorph Nectria
haematococca), Fusarium sp., Gibberella avenacea (anamorph Fusa-
rium avenaceum), Nectria mauritiicola, Nectria sp., Fusicolla sp. and
Davidiella macrospora (anamorph: Cladosporium iridis; Goetz and
Dugan, 2006). Endophytic species were rare; those encountered
were Acremonium strictum, Alternaria alternata, Chaetomium spp.,
Cladosporium malorum, Epicoccum nigrum, Geomyces vinaceus,
Geomyces luteus, Geomyces pannorum, Lewia infectoria (anamorph:
Alternaria infectoria and Alternaria alternata), Ulocladium sp., and
Pseudogymnoascus roseus. Some fungi encountered were mere
saprotrophs, i.e., Acremonium furcatum, Beauveria bassiana, Crypto-
coccus adeliensis, Mortierella alpina, Mortierella sarnyensis, Nectria
vilior, and Paecilomyces carneus.
The Simpson’s (1  D) and Shannon’s (H0 ) indices of diversity
revealed the largest level of culturable fungal diversity in the arable
soil planted with CDC Anna, whereas the lowest level of diversity
being endophytic were infrequently isolated, but they were diverse
(Supplementary material Table 4, Fig. 2). The abundance of
abundance of potential mycopathogens and phytopathogens,
whereas the abundance of potentially endophytic fungi was nega-
tively correlated with the abundance of potential mycopathogens
(Table 3).
The distribution of culturable fungi in topsoil planted with
chickpea was also related to chickpea genotypes, as revealed by
associated with the highest abundance of potential mycopath-
Potentially phytopathogenic taxa were relatively abundant in the
arable soil planted with CDC Anna, where they accounted for 4.3%
of all isolations, compared to 2.0% in that of Amit and 1.1% in soil
pathogenic fungal taxa, 6.1%, was found in CDC Frontier. The three
kabuli genotypes were principally associated with species of the
3.4. Diversity of AMF communities in topsoil planted with chickpea
A total of 15 AM fungal ribotypes were found at the study site
(Fig. 4). The leveling of all rarefaction curves (Supplementary
material Fig. 2) indicated that the intensity of clone sequencing
used in this study yielded a good representation of the AM fungal
diversity present in the soil planted with the different chickpea
genotypes. The arable soil planted with CDC Anna hosted the
 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141 135

Fig. 2. Profiles of the community of culturable fungi in the top 0e7.5 cm soil layer planted with the different chickpea genotypes in 2005. High frequencies of recovery from the
chickpea root zone due to soil dilution and plating are shown by lighter shades, whereas darker shades show low frequencies and black rectangles indicate undetected taxa.
Sequence identities, determined by comparison with known sequences, are followed by the percentages of similarity with the closest match in GenBank and preceded by their
accession number in GenBank. The functional group to which they belong is indicated in parentheses.

Table 2
Indices of diversity of the culturable fungal communities living in the top 0e7.5 cm Table 3
of soil planted with different chickpea genotypes in 2005. Relationship among the sizes of different functional groups of fungi isolated from
the top 0e7.5 cm of soil planted with chickpea in 2005.
Diversity index Chickpea genotypes
Variable Spearman coefficient of correlation
Amita CDC CDC CDC
Frontier Xena Anna Antagonistsa Dark Mycopathogens Phytopathogens
Simpson’s index of 0.828 b 0.881 ab 0.869 ab 0.003 a endophytes
diversity (1  D) Endophytes 0.2132ns
Shannon’s diversity 1.998 b 2.223 ab 2.192 ab 2.284 a Mycopathogens L0.3788* L0.3812*
Index (H0 ) Phytopathogens L0.4225* 0.0979ns 0.1039ns
a Saprotrophs and 0.2847ns 0.2510ns 0.2124ns 0.3393ns
Least square means are not significantly different according to ANOVA-
others
protected LSMeans Student’s t tests when followed by the same letter on the
same row (a ¼ 0.05 5; n ¼ 8). a
ns not significant; *significant at a ¼ 0.05 (N ¼ 36).
136 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141

Axis 2
CDC Xena

Penicillium-48

Bionectria ochroleuca-42
Bionectria ochroleuca-41
Penicillium sp.-49
Penicillium canscens-10
Penicillium sp.--15
Penicillium-67

Mortierella alpina-08

Axis 1
Hypocrea lixii-45
CDC Anna
Fusarium redolens-16 Amit
Hypocrea lixii-27
Bionectria ochroleuca-23 Mortierella alpina-68
Penicillium ochrochloron-09
Penicillium canescens-11
Trichoderm sp.-65
Penicillium aurantiogriseum-46
Gibberella avenacea-71 Bionectria ochroleuca-31

Penicillium canescens-58

CDC Frontier

Fig. 3. Relationship between the distribution of the culturable fungal species and chickpea genotypes, as revealed by canonical redundancy analysis (RDA) (P ¼ 0.016, N ¼ 36). Rare
species, i.e., those associated with less than two chickpea genotypes, were omitted. Numbers associated with fungal species names are the two last digits of their sequence accession
codes in GenBank, which begins with ‘JF3119’. Axes 1 and 2 explain 28.7% of the variance and are both significant at a ¼ 0.05 (N ¼ 36).

greatest richness of AM fungal ribotypes. In 2005, the genotypes
CDC Anna and Amit had greater (P ¼ 0.0001) root colonization than
the genotypes CDC Xena and CDC Frontier, but in the dry growing
season of 2006, all varieties were equally colonized (P ¼ 0.873)
(Table 1).
3.5. Durum wheat re-crop establishment
The soil legacy of the different chickpea genotypes influenced
the density of durum wheat plant stand only in 2009, as revealed by
the interaction of genotype
year (P ¼ 0.0437), in the ANOVA.

Fig. 4. Profiles of the AM fungal communities in the top 0e7.5 cm soil layer planted with different chickpea genotypes. Sequence identities, determined by comparison with known
sequences, are followed by the percentages of similarity with the closest match in GenBank and preceded by their accession number in GenBank. The distances between horizontal
lines in Ward’s dendrogram represent the amount of dissimilarity between the compositions of the AM fungal community profiles.
 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141 137

Durum wheat stands were denser following the desi chickpea CDC Table 4
Anna than following any kabuli chickpea (Fig. 5). Chickpea maturity date in 2008, 2009 and 2010, and water contents in the soil
profile at physiological maturity of these chickpea crops and before seeding wheat in
Summer 2008 received more precipitation than normal, 2009, 2010 and 2011, respectively.
whereas summer 2009 was hot and dry and 2010 was extremely
wet (Supplementary material Fig. 3). These climatic variations Average chickpea maturity date (Julian day)

affected the chickpea maturity date. Chickpea matured 11 days 2008 2009 2010
earlier in 2009 than in 2008 and 32 days later in 2010 than 2008 249 238 276
(Table 4). The soil moisture content was also lower after chickpea in Soil water after chickpea
2009 than in 2008 or 2010 (Table 4). Regression analysis revealed
2008a 2009 2010
that the density of durum wheat stand was positively correlated
0e15 2.07 b 1.67 c 2.78 a
with the amount of water contained in the 30e60 cm soil layer after 15e30 1.78 b 1.76 b 3.29 a
the chickpea crop in 2008 as well as in the following spring 30e60 3.18 b 3.46 b 5.91 a
(Table 5). The relationship between durum wheat plant density and 60e90 4.26 4.83 4.87
soil water content at a depth of 30e60 cm after the chickpea crop 90e120 5.51 5.78 5.35

was polynomial and existed only when less than 4 cm of water was Soil water before seeding wheat
present in this soil layer (Fig. 6). A weak negative correlation was 2009 2010 2011
also found between soil water content at the 30e60 cm depth after 0e15 2.98 b 2.97 b 3.68 a
the 2009 chickpea crop and the establishment of the 2010 durum 15e30 2.46 b 2.76 b 3.93 a
wheat crop (Table 5). 30e60 3.75 c 4.34 b 7.42 a
60e90 4.88 4.99 4.88
90e120 5.63 5.54 5.43
4. Discussion
a
Least square means are not significantly different according to ANOVA-
protected LSMeans Student’s t tests when followed by the same letter on the
Using plants to engineer the arable soil microbiome is possible. same row (a ¼ 0.05, n ¼ 16).
Our results and those of others (Berg and Smalla, 2009; Dunfield
and Germida, 2001; Micallef et al., 2009; Picard and Bosco, 2008;
microbiome, an ability that is lost when chickpea is subjected to
Schreiner et al., 2010) support this conclusion. Although our results
drought.
only offered a window on culturable fungi and a rough picture of
The discovery that genotypes of the same plant species could
the communities of active soil bacteria at the study sites, they show
configure soil microbial communities and induce disease-
that different living plants have different selective influences on the
suppressiveness (Mazzola, 2004; Mazzola and Gu, 2000) revealed
taxonomic and functional structure of the soil microbial commu-
the possibility of selecting plants that are able to create a soil mi-
nity. Different plants can produce different types and amounts of
crobial environment conducive to plant health and productivity
root exudates and use different amounts of soil resources, thus
(Marschner et al., 2005; Ryan et al., 2009; Wissuwa et al., 2009).
acting as important selective forces that shape the soil microbiome.
Intraspecific variation in the quality of plants as hosts for beneficial
In addition, our results show that the water stored at 30 cm and
microorganisms, a necessary condition for plant breeding, was
deeper in the profile of a soil planted with chickpea is a determi-
found in several crop plant species, including wheat (Hetrick et al.,
nant of the structure and composition of microbial communities
1992; Singh et al., 2012), rice (Engelhard et al., 2000), maize
living in the surface soil and of the soil legacy of chickpea crops for
(Cheeke et al., 2012; Kaeppler et al., 2000), soybeans (Khalil et al.,
the subsequent crop. This long-distance relationship between the
1994), and peanuts (Quilambo et al., 2005). We recently showed
soil’s water content at a depth of 30 cm or more and the microbial
that different chickpea genotypes vary in their profiles of bioactive
community in the surface soil underlines the importance of the
phytochemicals (Cruz et al., 2012; Ellouze et al., 2012) and are
plant as a determining influence in the arable soil microbiome, with
associated with different rhizobacterial communities (Yang et al.,
possible consequences for the next crop in rotation. The data show
2012b). We have now demonstrated the variation in the influence
that soil water reserves 30 cm below the soil surface are an
of chickpea genotypes on the arable soil microbiome.
important resource that allow chickpea to modify the arable soil
Previous research has demonstrated the feasibility of improving
the health of cropping systems through the use of certain crops
(Larkin and Griffin, 2007; Larkin, 2008, 2010, 2011a, 2011b) and

Table 5
Level of significance (P values) of the relationships between durum wheat plant
density and soil water contents at 0e15, 15e30, 30e60, 60e90, 90e120 cm depths at
maturity of the chickpea crops and before seeding durum wheat on the same soil in
the following spring, from 2008 to 2011.

2008e09 2009e10 2010e11

After chickpea harvest in fall
0e15 0.166 0.125 0.611
15e30 0.171 0.752 0.947
30e60 <0.001** L0.041* 0.955
60e90 0.315 0.957 0.819
90e120 0.231 0.658 0.776
Before seeding durum wheat in spring
0e15 0.626 0.570 0.287
15e30 0.524 0.828 0.764
Fig. 5. Density of the durum wheat plant stands established in 2009, 2010, and 2011 in
30e60 0.023* 0.760 0.119
plots planted with different genotypes of chickpea in 2008, 2009 and 2010, respec-
60e90 0.843 0.804 0.848
tively. Bars represent least square means that are significantly different when associ-
90e120 0.117 0.876 0.448
ated with different letters, according to ANOVA-protected LSMeans Student’s t
(a ¼ 0.05; n ¼ 4). *Significant at a ¼ 0.05 level; **significant at a ¼ 0.01 level (n ¼ 4).
138 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141

190 190

Durum stand density (Plant m2)

Durum stand density (Plant m2)
180 180

170 170

160 160

150 150

2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 2.5 3 3.5 4

Water content (cm) Water content (cm)

Fig. 6. Relationship between the density of the durum wheat plant stands and the content of water in the 30e60 cm soil layer after the chickpea crops in 2008 and prior to seeding
the durum wheat crops in the same soil in 2009 (n ¼ 16).

certain crop genotypes (Hopkins et al., 1987; Larkin et al., 1993; in particular F. solani (teleomorph N. haematococca), F. oxysporum,
Mazzola and Gu, 2000). Genetic variation in chickpeas reveals the F. redolens, and G. avenacea (anamorph F. avenaceum). As these
possibility of selecting genotypes with positive effects on the soil species are known to infect the core components of the cropping
biotic environment that are conducive to plant health. The traits to systems of the semiarid grasslands of North America, i.e., wheat,
select for remain open questions at this time. Different mechanisms durum wheat, lentil, pea and chickpea (Esmaeili Taheri et al., 2011;
may lead to disease suppressiveness in soil and enhanced plant Holzgang and Pearse, 2000), it appears that the positive effect of CDC
performance. Mycoparasitism, antibiotic production, induction of Anna on the biotic properties of soils is most likely suboptimal and
plant defense mechanisms by certain microorganisms, and could be improved.
competition with pathogens for resources, mainly soil carbon, can The identity of the taxa that make up plant growth-promoting
all suppress the activity of soil-borne pathogens (Shennan, 2008; or disease-suppressive communities and consortia may be as
Weller et al., 2002). Soil disease suppressiveness can be specific and important as richness, as shown previously (Mendes et al., 2011;
involve the effects of a few microorganisms, or it can be general and Wehner et al., 2010). Richness may not be sufficient to predict soil
attributable to the total soil microbial biomass (Weller et al., 2002). health, but it may be a prerequisite to soil health. Certain Tricho-
The positive influence on the establishment of a subsequent crop of derma were associated with soil suppressiveness to take-all of
wheat that was observed after CDC Anna, the chickpea genotype wheat (Duffy et al., 1996; Dunlop et al., 1989), and the involvement
associated with the highest level of soil microbial diversity, sup- of non-pathogenic F. oxysporum in the suppression of Fusarium
ports the notion that biodiversity is an important attribute of wilts in suppressive soils is well documented (Larkin and Fravel,
productive soils. 1999; Larkin et al., 1996). Species of Trichoderma and the tele-
The examination of the disease-suppressiveness of soil reported omorph of T. harzianum, Hypocrea lixii, were commonly found at
for the semiarid zone of the Great Plains of North America revealed our site, especially in the arable soil planted with CDC Anna. Bio-
mechanisms of specific suppressiveness attributable to two cul- nectria, another mycopathogen-hosting taxon that was diverse and
turable dark fungal endophytes (Andrade et al., 1994) identified as abundant at our site, is renowned in the field of biocontrol
Phialophora spp. (Zriba et al., 1999). The involvement of this genus (Hermosa et al., 2010; Sharma et al., 2011) and may represent an
in the suppression of take-all in North America (Zriba et al., 1999) additional resource for the reduction of disease in cropping sys-
concurs with previous reports from the grasslands of the UK tems. F. oxysporum was rare but was encountered in the rooting
(Deacon, 1973, 1976). zone of CDC Anna. The pathogenicity status of this F. oxysporum is
Dark endophytic fungal species were relatively rarely detected unknown. The involvement of non-pathogenic isolates of the
in arable soil planted with chickpeas in our study, providing potentially phytopathogenic species of F. oxysporum underlines the
insufficient data to confirm the role of dark endophytes in complexity of the interactions taking place in the soil.
improving soil health through competition but not ruling out this The AM fungi benefit plants by reducing the incidence of soil-
possibility. Dark fungal endophytes can enhance the production of borne plant diseases (Lioussanne et al., 2008; Wehner et al.,
plant growth by promoting metabolites and antibiotics toxic to 2010). They also mitigate the impact of cold (Paradis et al., 1995)
Oomycetes (Tellenbach et al., 2013). These fungi, are known as K- and drought by strengthening the physiological response of plants
strategists with slow growth rate and poor sporulation (Rodriguez (Al-Karaki et al., 2004; Aroca et al., 2008; Porcel et al., 2006, 2003;
et al., 2009) and cultural methods, as used here, yield a better Ruiz-Lozano et al., 2001). The AM fungi improve plant uptake of
representation of heavy sporulating species than poor sporulators. nutrients (Atul-Nayyar et al., 2009; Hodge and Fitter, 2010; Liu
Higher levels of diversity are expected to lead to greater pro- et al., 2007), contributing to plant health, whereas their abundant
ductivity and stability of agro-ecosystems (Chaparro et al., 2012; hyphal networks (Leake et al., 2004) improve soil structure (Augé,
Shennan, 2008). The community of culturable fungi and AM fungi 2004; Six et al., 2004) and water infiltration and retention. The
created under the influence of CDC Anna had the highest levels of high level of AM fungal richness in the arable soil planted with CDC
richness and evenness of all genotypes examined in our study, and Anna in this study suggests that this chickpea genotype is a good
the abundance of FAME markers in the arable soil planted with this host for AM fungi and may benefit a subsequent crop that is
genotype was high, indicating the presence of an abundant bacterial responsive to AM symbiosis.
community. The soil legacy of CDC Anna positively influenced the The effects of genotypes on the soil microbiota observed in 2005,
establishment of the subsequent durum wheat crop, compared to all a normal growing season at the study site, were overridden by the
kabuli chickpeas in our re-crop experiment (Fig. 4), demonstrating effects of a drought in 2006. Drought can affect the structure of the
that healthy soils are biodiverse. However, CDC Anna was associated rhizobacterial community of chickpeas much more sharply than
with a relatively high proportion of potentially pathogenic species, genotype (Yang et al., 2012a). The results of the durum wheat re-
 W. Ellouze et al. / Soil Biology & Biochemistry 63 (2013) 129e141
crop experiment also suggest that plants influence the soil micro-
biota only in conditions of adequate soil moisture. Controlled-
condition experiments are required to define the conditions for
expression and the mechanisms involved in the effects of the
chickpea genotype on the soil microbiota. Our observation of a
relationship between the expression of chickpea effect on the
composition of soil microbial communities living in topsoil and
water stored below 30 cm in the soil profile suggests that the effect
of plants on the soil microbiota is modulated by the resilience of the
plant to water shortage and excess. Extreme dryness and precipi-
tation events also directly affect soil microorganisms (Hamel et al.,
2006; Manzoni et al., 2012; Yang et al., 2012a). Extreme soil water
conditions, particularly drought, appear to limit the benefits of
engineering crop biotic environments through the use of selected
plant genotypes in rainfed crops of semiarid regions.
4.1. Conclusions
Our study demonstrates that the diversity of soil bacteria and
fungi can be influenced by plant genotype, which suggests the
possibility of increasing the sustainability of cropping systems
through the use of selected genotypes that create soil biotic con-
ditions conducive to plant health. Our observations support the
notion that microbial diversity is an important component of pro-
ductive soils. The repression of phytopathogenic fungal species that
is expected in disease-suppressive soil environments is often
attributed to one or a few antagonists, but in this study, plant
growth promotion was achieved as the soil legacy of the chickpea
genotype increased the soil microbial diversity levels. We reveal the
identity of several potential fungal allies of crop plants that can be
selected by chickpea genotypes in the semiarid soils of the North
American plains and show how the influence of plants on the soil
microbiome could collapse with extreme rainfall or periods of
drought. This study demonstrates the feasibility of engineering the
arable soil microbiome through the use of selected genotypes and
identifies climatic fluctuations as a possible limiting factor in
rainfed cropping systems of semiarid grasslands of North America.
Acknowledgments
The authors would like to gratefully acknowledge the excellent
comments of two anonymous reviewers and the technical assis-
tance of Keith Hanson, Cal McDonald, and Lee Poppy. We also thank
Stéphane Daigle for his guidance in the statistical analyses. This
project was supported by a grant from the Alberta Pulse Growers
and Agriculture and Agri-Food Canada to Hamel and Gan, a Natural
Resources and Engineering Research Council of Canada Discovery
Grant to St-Arnaud and a scholarship to Ellouze from the Islamic
Development Bank Merit Scholarship Programme for High
Technology.
Appendix A. Supplementary material
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.soilbio.2013.04.001.
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