Experiment 1: rhizotron growing systems and sample collection
for microbial analysis
A rhizotron system (30 cm long × 18 cm wide × 2 cm thick) with a
removable transparent acrylic side was designed for the current
experiments. This system was modified from DeAngelis et al (2009) who
confirmed its effectiveness to analyse rhizosphere microbiome by keeping
healthy plants and avoiding bacterial contamination. Rhizotrons were
covered with aluminium foil to simulate dark conditions for root growth,
and angled at 45 degrees, with the transparent side as the base, to
enable visualisation of roots under the transparent surface (Fig. S1).
Five soil types and three chickpea genotypes with 4 replicates were
examined in this rhizotron system. Five soils were collected from the
cropping regions of South Australia, Australia, and marked as Soil-A
(-34.511, 138.697), Soil-B (-34.509, 138.667) and Soil-C (-34.537,
138.690), Soil-D (-34.967, 138.633) and Soil-E (-34.966, 138.631). All
the soils used here were from typical Mediterranean type dryland farming
systems with 3 to 4 years of cereal crops (wheat or barley) rotated with
one season legume (lentil, field pea or faba bean). To limit the
excessive presence of Mesorhizobium ciceri (the symbiotic
rhizobia of chickpea), soils were chosen where no chickpea was planted
in the last decade. The soils differed in chemical and physical
properties (Table S1), which enabled the evaluation of stochastic or
deterministic processes of host plants in assembling microbiome at
different niches. Soil physiochemical properties including total N, C,
P, K, Ca, Zn, Mg, Mn and Cu, pH, EC, and texture were analysed using the
protocol from Rayment & Lyons (2011), and the results are presented in
Table S1. Chickpea cultivars Genesis090, PBA Slasher and Sonali were
used. Previous evaluation across multiple environments with diverse
photoperiod, seasonal rainfall, and cropping management during chickpea
growth (Sadras et al., 2016; Kaloki et al., 2019) have demonstrated that
Sonali (genotype EF) required less thermal time to flowering compared
with the late flowering genotypes Genesis090 (genotype LF1) and Slasher
(genotype LF2). It has been confirmed that an 11-bp deletion in chickpea
early flowering 1 (Efl1 ) locus was associated with early
flowering performance (Ridge et al., 2017). We analysed the structure ofEfl1 gene by sequencing the whole genomes of the three cultivars
in the present study. DNA sequencing and mapping were described in (Li
et al., 2017). Briefly, genomic DNA of Genesis090, PBA Slasher, and
Sonali was extracted from young leaf tissue using Qiagen DNeasy Plant
Mini Kit. Pair-end reads (100 bp) of each genotype were sequenced using
Illumina HiSeq 2000 platform. Sequencing reads were filtered, trimmed,
and mapped to the reference genome 2.6.3 (http://cicer.info) using
SOAP2. Bam files were filtered out for reads with more than 3 mismatches
resulting in ~10 x coverage of each genome. Bam files
were imported into the software Integrated Genome Viewer (IGV) for
visual inspection of the Efl1 gene.
Chickpea seeds were surface-sterilized by soaking in 10% sodium
hypochlorite for 10 min, and were then washed with sterile water three
times, soaked in 70% ethanol for 20s, and then washed another three
times in sterile water. Surface-sterilised seeds were germinated in a
petri dish with autoclaved sand and water until the root emerged to a
length of 0.5 mm. Two germinated seeds were transplanted to 2 cm depth
in each sterilised rhizotron that had been filled with soil at a
consistent bulk density of 1.38 Mg m-3. Rhizotrons
without plants were used as the bulk soil control.
Plants were grown in a controlled environment room (800 μmol
m-2 s-1 light for 12 hours at 20 °C
and 12 hours of darkness at 10 °C), and watered to field capacity every
two days by weighing using autoclaved sterilised water. The point of
observable root tips was marked each day. At 16 days after planting
(DAP), roots with rhizosphere soil were harvested. Root segments between
the markers of 1 to 2 DAP and 15 to 16 DAP were cut off with
approximately 1.0 mm of soil adhering around the root; these were
regarded as the basal and apical zones, respectively. Root segments from
two plants in each rhizotron were processed for microbial community
analysis. Rhizosphere and endosphere samples were isolated from root
sections using a method modified from Bulgarelli et al. (2012). The
sampled root segments with attached soil were transferred into 15 mL
sterile PBS buffer in a 50 mL Falcon tube, which was placed on an
orbital shaker for 20 min and rotated at 180 rpm. Roots were collected
using sterilised tweezers for further processing. The rest of the soil
suspension was centrifuged at 180 rpm for 20 min. After removing the
supernatant, the pellet was defined as rhizosphere soil. Roots were
washed again on the shaker for 20 min at 180 rpm, and then transferred
into a new 50 mL Falcon tube with 40 mL sterile PBS buffer. The roots in
the Falcon tube were sonicated for 10 cycles of 30 s at 43 kHz and 120 W
(model 160HD, Soniclean, Australia) with a 30 s break. The microbiome
extracted from the sonicated root was considered to be from the
endosphere. The rhizosphere and endosphere samples were stored in a -80
°C freezer.
The total DNA of the rhizosphere soil, cleaned root tissue, and bulk
soil was extracted using a PowerSoil DNA isolation kit (Mo Bio,
Carlsbad, CA, USA) based on the manufacturer’s instructions. Afterwards,
the V3-V4 region of 16S rRNA genes was amplified using the forward
primer 341F (CCTAYGGGRBGCASCAG) and reverse primer 806R
(GGACTACNNGGGTATCTAAT). The PCR products were sequenced by Australian
Genome Research Facility on an Illumina MiSeq platform with 300 bp
paired-end reads following the manufacturer’s instructions.
Quantitative PCR (qPCR) was performed to determine the quantity of
bacterial DNA in the samples, using the FemtoTMBacterial DNA Quantification Kit (Zymo Research, USA) following the
manufacturer’s instructions. The universal bacterial 16S rRNA gene was
amplified using the Femto Bacterial qPCR premix on the CFX connect
Real-Time PCR detection system (Bio-Rad, USA), as follows: initial
denaturation at 95°C for 10 min, 40 cycles of 95°C for 30 s, 50°C for 30
s and 72°C for 60 s, and final elongation at 72°C for 7 min. Gene copy
numbers were estimated using E. coli strain JM109 as an internal
standard. Three technical replicates per sample were analysed. Data were
calculated as copy number per gram of the soil or root tissue used for
DNA extraction.