To assess the bacterial communities associated with the rhizospheres of both vascular plants, 5 g of the roots with aggregated soil was shaken in a saline solution (NaCl 0.85%) for 2 hours. Then 50 mL of the supernatant was centrifuged for 10 minutes at 9,000 rpm, and 0.5 g of the pelleted soil was used for DNA extraction. For the bulk soil, 0.5g of each sample was used for the extractions
Growth protocol
The study used a non culturable method to access the bacterial communities in soils and rhizospheres
Extracted molecule
genomic DNA
Extraction protocol
The DNA extractions were made using the Fast DNA Spin Kit for soil (QBIOgene, Carlsbad, CA) following the manufacturer’s instructions.
Label
Cy5
Label protocol
For the microarray assays all the triplicate rhizosphere and bulk soil samples from the six sample sites were used (47 samples). A 16S rRNA gene fragment was amplified using the universal primers F1-R13 (Lane 1991). The PCR mixture comprised 1 µl of template DNA (10 - 20 ng), 20 pmol of universal primers, 5 µl of 10 x PCR buffer (Fermentas, Burlington, Ontario, CA), 1.5 mM MgCl2, 2.5 U of Taq DNA polymerase (Fermentas), 0.2 mM of each dNTP and sterile filtered water to a final volume of 50 µl. The temperature profile included an initial denaturation step at 94°C for 5 min, 35 cycles of a denaturation step at 94°C for 30s, a primer annealing step at 50°C for 30s and an extension step at 72°C for 45s, followed by a final step of 7 min at 72°C. PCR products were purified using the PureLink TM PCR purification Kit (Invitrogen Canada, Burlington, Ontario, CA).
Hybridization protocol
Slides were pre-hybridized for 1 h at 37°C with a DIG easy hybridization solution (Roche Applied Science, Laval, QC, Canada) containing 5% BSA. Samples were then assigned randomly to a sub-array and hybridization was carried out for approximately 20 h at 37°C on a Slide Booster apparatus (Implen, Calabasas, CA). Slides were washed three times for 5 minutes at 37°C in 0.1X SSC / 0.1% SDS followed by a single wash for 5 minutes at 37°C in 0.1X SSC.
Scan protocol
Slides were scanned in a ScanArray GX PLUS Microarray Scanner (Perkin-Elmer, Boston, MA) at a resolution of 10 µm.
Data processing
Images were transferred to QuantArray (Perkin-Elmer) where each spot was identified and quantified. Data was then exported to Excel where net spot intensity was calculated by subtracting the background signal from the target spot intensity. A spot was scored as “present” if its net intensity was at least 3 times higher than the median net intensity of all spots. For a taxon to be scored as present, all of its triplicate spots had to be scored as present. The results from the 16S rRNA gene microarray were only used in the binary form (presence/absence of taxa).
