Experiment 2: root exudates collection and analysis
For the root exudate collection, three chickpea genotypes were planted
in the rhizotrons as described in Experiment 1 with 4 replicates.
Rhizotron system with soils (only Soil-C used) was sterilised by 25k
Gray gamma radiation, as soil microbes can degrade the root exudates
rapidly. The growing condition and management were the same as
Experiment 1. Sample preparation for root exudate analysis was based on
a method modified from Wouterlood et al. (2004) and Zhou et al. (2020).
The attached soil on sampled apical and basal segments of roots were
transferred into 50 mL Falcon tube with 10 mL deionized water, and then
microwaved by a 700W microwave oven for 10 s to prevent metabolic
activities (Zhou et al. 2020). The sample was washed on an orbital
shaker for 30 min at 180 rpm and the root was then discarded. The soil
suspension was filtered through Whatman no. 42 filter paper. The
filtered solution was transferred to a syringe filter (0.1 um), and then
stored at -80 °C for further analysis. The soil collected was dried at
105 °C to measure soil dry weight. The bulk soil was collected as the
control, and processed using the same method as the rhizosphere soil. To
check if there was bacterial contamination in the sterilised system, the
sampled rhizosphere and bulk soil were diluted at 10-1using sterile MilliQ water, then 100 μL soil suspensions from the
dilutions were spread onto Petri dishes containing 15 mL of 10%
tryptone soya agar (TSA). Plates with parafilm were incubated at 28°C
for 5 days, and no bacteria colonies were observed to conform the
sterilised condition of the root exudation collection system.
Root exudates were analysed for carbohydrates and amino acids at the
Centre for Carbon, Water and Food in The University of Sydney, following
the method in Conselvan et al. (2018). Carbohydrate and sugar analysis
were measured by gas chromatography (GC). The analysis was carried out
on an Agilent 6890 Gas Chromatograph with QQQ 7000 Mass selective
detector with a HP5 column (0.25 mm i.d., 30 m, 0.25 mm film thickness;
Agilent Technologies, Santa Clara, United States). Split injection was
made at 300 °C with an initial oven temperature program of 60 °C for 2
min, ramping to 300 °C at a rate of 10 °C min-1 and
maintained for 10 min. Column flow rate was maintained at 1.5 mL
min-1. Mass Hunter software (version B.07.01, Agilent
Technologies) was used for peak integration. For amino acid analysis,
LC-MS analysis of the extract was carried out on a 1290 Infinity LC
system coupled to a 6520 QTOF Mass selective detector (Agilent
Technologies, Santa Clara, United States). A 3.5 uL sample was injected
into a Zorbax SB-C18 column (2.1 × 150 mm, 3.5 µm) and separation was
achieved by gradient elution with water and methanol. The QTOF was tuned
to operate at the low mass range <1700 AMU and data
acquisition was performed in scan mode (60-1000 m/z) and ionization was
set to positive ion mode. LC-MS results were identified based on their
retention times relative to standards as well as their formula mass.
Peaks were integrated and their relative quantities were calculated
using the Mass Hunter software. The measured compounds were estimated
relative to the amount of dry soil and growing period (1 d for apical
root and 15 d for basal root). Root exudate was calculated as the
difference in measured compounds between rhizosphere soil and bulk soil
(Zhou et al. 2020).