DNA extraction, sequencing and bioinformatics
We prepared DNA extractions from T. triandra root endospheres
following methods outlined in Hodgson et al. (2024b). This
involved cleaning the exterior of plant root surfaces by sonication at
five 30 s on/off burst cycles in 0.02% Silwet L-77 supplemented PBS
buffer (pH = 6.5) for 5 min, followed by five 5 min washes in
sterilised, distilled water. These methods underwent prior validation
described in Hodgson et al. (2024b). To extract microbial DNA
from rhizospheres, we followed the protocol from McPherson et al. (2018). Briefly, root samples were washed in 0.02% Silwet L-77
supplemented PBS buffer, vortexed for 45 min and then filtered using 100
µm sieves (Thermo Fisher Scientific, Waltham, USA) and centrifuged at
1000 RPM. Soils samples taken from the plant plots at either the start
or end of the trial were stored at -20 ˚C after collection. DNA from
soil, rhizosphere and endosphere samples was extracted using the DNeasy
PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany), as per
manufacturer’s instructions.
Amplicon libraries of the 16S rRNA V3-4 gene region were generated by
the Australian Genome Research Facility (Brisbane, Australia). Sequences
were generated using the 300 base pair paired end run of the Illumina
NextSeq 2000 platform. The DADA2 bioinformatics pipeline was used to
infer amplicon sequence variants (ASVs), with monotonicity enforced
during error estimation. Additionally, loess smoothing was applied with
specified weights, span, and degree to improve error rate modelling.
Qiime2 was used to identity profiles from amplicon sequence data from
the SILVA database (v138.1) (Wang et al. 2007; Quast et
al. 2013), using a naïve Bayesian classifier (Wang et al. 2007;
Callahan et al. 2016; Bokulich et al. 2018). Taxa that
were not assigned as Bacteria, unassigned at the Phylum level, and
associated to mitochondria or chloroplasts were removed. We were unable
to extract and sequence viable DNA concentrations from sterilised low
aridity conditions at the beginning of the experiment, possibly due to
the sterilising effects of autoclaving on microbiota and their DNA.