Methods
\label{methods}
Studied sites and soil collection
(JT?)
\label{studied-sites-and-soil-collection-jt}
The two studied sites are located in the central-West area of Belgium at
Gaurain-Ramecroix (coordinates?) and Wiers (region of Tournai). The
region is characterized by an oceanic temperate climate with an average
annual rainfall of 800 mm and an average annual temperature of 10°C
(IRM, Begium). According to the WRB-FAO, the soil type of the both sites
is classified as Endogleyic Retisol. The two sites are characterized by
contrasting pedological conditions explained by different soil textures:
Fine Sandy Loam (so-called “Sandy site”) and Silt Loam (so-called
“Silty site”).. Within each site, we selected three pairs of field,
each pairs being composed of two farming systems: organic versus
conventional. . The soil samples were collected in three replicates,
each replicates being a composite of 6 sub-samples collected from the
surface horizon (0–20 cm) in July 2015 (before harvest).
Farming system description
(Fanny?)
\label{farming-system-description-fanny}
The organic fields (at the both site) are managed by only one farmer,
while the conventional fields are managed by different farmers.
At the “Sandy” site, fields under organic farming were intensively and
conventionally managed until 1995 including a deep plowing (30 cm) and
the use of chemical inputs (fertilizers, fungicides, herbicides and
insecticides). Since 1992, the organic farmer decided to gradually
replace intensive plowing by minimum tillage (3 to 5 cm) and finally
direct seeding with zero tillage in 2002. In 2011, the use of chemical
input was stopped and in 2015 the farm obtained the official status of
“organic farming”. At the “Silty” site, the history of farming until
2010 is unknown, but since this period the fields are managed in the
same way as for the “Sandy” site: direct seeding and no chemical
inputs. The organic farming is continuously covered by a mulch made up
of a mix of leguminous and ? A compléter!!!!
Soil chemical properties
\label{soil-chemical-properties}
The soil samples were dried at 40°C and sieved to <2mm. The
water content was measured by weighing the sample before and after
drying it at 105°C according to ISO 11465 (1993). Total C and total N
contents were measured by dry combustion (ISO 10694, 1995) under the
combined action of elevated temperature (more than 900°C) and oxygen
flow. CO2 produced is separated by gas chromatography. Given the absence
of carbonates in the soil samples, the total C measured corresponds to
total organic C. Exchangeable cations
(Ca,
Mg,
K and
Na) and P extraction was
carried out with the modified Metson method (Pansu & Gautheyrou, 2006)
and their concentrations were determined by atomic absorption
spectroscopy and P concentration was determined by spectrophotometry.
Cation-exchange capacity (CEC) was measured using the cobaltihexamine
chloride method according to ISO 23470 (2007). Determination of
pH(water) and pH(KCl) were carried out by using a soil/solution (water
or KCl 1M) ratio of 1:5 (ISO 10390, 2005). Particle size distribution
was determined by sedimentation using the pipette method.
Soil biological
properties
\label{soil-biological-properties}
High-throughput sequencing of bacterial and fungal
ribosomal
markers
\label{high-throughput-sequencing-of-bacterial-and-fungal-ribosomal-markers}
DNA was isolated from the soil samples (8 g wet weight) with the
PowerMax® soil DNA isolation kit (MO BIO Laboratories, Solana Beach, CA)
according to the manufacturer’s recommendations.
Amplicon generation (bacterial V3-F4 and fungal ITS2 of the ribosomal
RNA operon) was performed as described by Frey et al. (2016). The
amplicons were sent to the Génome Québec Innovation Center at McGill
University (Montréal, Canada) for barcoding and paired-end sequencing on
the Illumina MiSeq V3 platform (Illumina Inc., San Diego, CA, USA). The
sequence data were denoised according to Frey et al. (2016), including
paired-end read assembly (REF), elimination of sequencing errors (REF)
and chimeras (Edgar, 2013), as well as target verification and
extraction (Hartmann et al., 2010; Nilsson et al., 2010). Denoised
sequences were clustered into operational taxonomic units (OTUs) at 97%
of identity and queried against Greengenes (DeSantis et al., 2006;
McDonal et al., 2011) and UNITE (Abarenkov et. al, 2010) using the naive
Bayesian classifier (Wang et al. 2007) and a minimum bootstrap support
of 60%.
Statistics
\label{statistics}
The experimental design was as follow: The sites (sandy and
silty ) and farming systems (organic and
conventional ) were considered as fixed factors, and the spatial
component (plot) was considered as random factor nested within
the site factor (plots 1 to 3 at sandy site and 4 to 6 at
silty site).
Estimates of alpha-diversity, i.e. richness Sobs and Smith-Wilson
evenness E (Smith and Wilson, 1996), were based on evenly rarefied OTU
abundance matrix and computed in mothur. The significance of the factors
effect on alpha-diversity was examined using univariate Permanova based
on Euclidean distance. Differences in beta-diversity were detected using
the Bray-Curtis ecological distance calculated on the basis of
normalized and square root transformed OTU abundance and after rare OTUs
(singletons) were removed. The major variance components of bacterial
and fungal beta-diversity was examined using principal coordinate
analyses (PCO). The hypothesis were tested using the Permanova routine
(Anderson, 2001) implemented in the software Primer6+ (Clarke and
Gorley, 2006) with 99999 permutations. The presence of a priori
groups with reference to the hypothesis was visualized with constrained
analysis (CAP). The significance of the test was determined based on the
Monte-Carlo empirical P value. The positive or negative response of taxa
to farming system for each site was identified using the function adonis
with 9999 permutations on centered and scaled OTUs relative abundance.
Rare OTUs (<0.01% and those that occurred in less than 3
samples) were removed. Adjustments for multiple testing were performed
using the Benjamini-Hochberg correction (Benjamini and Hochberg, 1995).