Old Eucalyptus Time 4 Replicate 2
Collected soil samples were removed from liquid nitrogen and thawed on ice until nucleic acids extraction. Around 500 mg of soil from each sample was used to DNA extraction, using the Fast DNA Spin Kit for Soil (MP Biomedicals, USA), and purified using the NucleoSpin Soil Kit (Macherey-Nagel, Germany) from its sixth protocol step, due to the dark color after the final step of extraction. Time 4 soil samples were sequenced by Star Seq company (www.starseq.com, Germany) on a MiSeq equipment, using paired-end runs (2 x 250), according to manufacturers guidelines. The primers used were the 515FB (GTG YCA GCM GCC GCG GTA A) and the 926R (CCG YCA ATT YMT TTR AGT TT), that assess the V4-V5 regions of the 16S rRNA gene. Bioinformatic analysis were done using the Mothur software (v. 1.41.3; Schloss et al., 2009).