Large variability of soil microbial diversity and functions in an over 20-year old Eucalyptus grandis plantation
The study was conducted in the south of Brazil, at the experimental station of forest sciences of Itatinga (ESALQ, University of São Paulo), which has a tropical humid climate. The soils are deep Ferralsols (F.A.O., 1998), and their mineralogy and physico-chemical characteristics are detailed in Maquere et al. (2008). The site was planted in 1998 with Eucalyptus grandis at a tree density of 1666 individual trees ha-1. Last fertilization at the site occurred in 2008, with no management for the last 10 years, meaning that the understory vegetation was allowed to develop. A 198 x 276 m stand of rather homogeneous topography was defined and divided in 108 experimental plots of 20 m x 23 m each. Plots were distributed in three blocks separated by a 9 m wide interrow (the blocks were not used in the present study). To account for the natural heterogeneity of this area, a sampling was done in March 2019 (during the dry season). The 10 cm-depth surface soil was collected using an auger in a total of 54 plots spread throughout the stand (data could be processed for 51 plots only). Each sample was a mixture of 9 soil cores taken in the plot (with three cores yielded in three inter-rows in the center of the plot). All samples were sieved at 2 mm and immediately frozen or air-dried for storage before molecular and other analyses, respectively.  In order to characterize bacterial and fungal communities, total soil genomic DNA was extracted from 500 mg of soil with the FastDNA SPIN™ kit for soil (MP Biomedicals Santa Ana, CA, USA) as described by Tournier et al. (2015). Extracted DNA was done using PicoGreen fluorescence (Molecular Probes, Paris, France) and kept at -20 °C until amplification. Initial DNA quantity (2.5 ng) was amplified in a total reaction volume of 10 µl containing 1X of Sso advanced SYBR Green supermix (BioRad, Hercules, CA, USA) and 0.5 uM of each primer. qPCR assays were run in duplicate using a Biorad CFX96 Real-time PCR System and the results were analyzed with the Software Bio-Rad CFX Manager 2.0. Bacterial and fungal community diversity were assessed by amplifying the V3-V4 region of the 16S rRNA and the internal transcribed spacer ITS2 of the nuclear ribosomal RNA.  To improve recovery and limit PCR bias, three PCR replicates per sample were pooled. All amplicon products were analyzed using paired-end Illumina MiSeq sequencing.