How deep ectomycorrhizae can go? A case study down to 4-meters in a Brazilian eucalyptus plantation

The experiment was conducted in the Itatinga research station of the University of São Paulo in Brazil (23°02′S, 48°38′W). The climate is subtropical humid with a mean annual rainfall of 1360 mm and a mean temperature of 19°C (with a seasonal cold and dry period between June and September). The soil is a deep Ferralsol developed on Cretaceous sandstone. In brief, this is a highly weathered soil, dominated by the sand fraction, with a clay content ranging from 15% in the top soil to 20-25% in deep soil layers. Soil pH is acidic and nutrient contents are low.A split-plot experimental design was set up in 2010 with a highly productive Eucalyptus grandis clone (provided by the Suzano Company, Brazil). In brief, six treatments (three fertilization regimes x two water regimes) were applied in three blocks. The area of individual subplots was 864 m2, with 144 trees at a spacing of 2 x 3 m. Our study was carried out for one modality of fertilization (non-limiting tree growth) representative of the silviculture in commercial eucalypt plantations (12 g N m-2, 3.3 g P m-2, 17.5 g K m-2, 200 g m-2 of dolomitic lime and trace elements applied the first 3 months after planting). Two pits were sampled between May and June 2015, in the same block, one in the ‘exclusion' plot (W-) and the other pit in the ‘exclusion-free' plot (W+). W- plots were equipped with two 40-cm wide plastic sheets in each inter-row to exclude 37% of the throughfall. The sampled trees were 5-year-old with a mean tree height of 20-m and root front depth down to 17-m depth. A 1.5-m wide square pit was dug in the 3-m wide inter-row of both the exclusion (W-) and control (W+) plots. Fine roots (< 2 mm) and ECM root tips were manually and very carefully collected from the soil, layer per layer, during pit digging in order to prevent any contamination between two consecutive soil depths and to collect the maximum biomass of fine roots in each soil layer. Ten soil layers were sampled from 0 to 400 cm depth (at 0-20, 20-40, 40-60, 60-100, 100-150, 150-200, 200-250, 250-300, 300-350, and 350-400 cm). Fine roots and ECM root tips were washed with tap water and stored at -20°C until analyses. Before performing molecular analyses, all the samples were ground with liquid nitrogen.DNA was extracted from 80 mg of fine roots or ECM root tips by using the FastDNA SPIN KIT (MP Biomedicals Santa Ana, CA, USA) according to the manufacturer's recommendations with some modifications. Briefly, samples were homogenized in 800 µl of CLS-VF buffer and 200 µl of PPS with a vortex step during 10 min, followed by an incubation of 15 min at room temperature.The samples were centrifuged (14 000 g, 10 min), and a binding matrix volume equal to the volume of supernatant was added, and agitated during 5 min at room temperature. After a centrifugation (14 000g, 10 min), a guanidine step washing was added as described by Tournier (2015). The DNA Binding Matrix was resuspended in 500 µL of guanidine thiocyanate (5.5 M),transfered to a Spin Filter column and centrifugated at 14 000 g for 1 min. This step was repeated and followed by two washing steps with SEWS-M. Finally, the DNA was eluted in 100 µl of DES. For all samples, extractions were made in triplicate and stored at -20°C before further analyses.The Internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal RNA were amplified using the primers ITS1FI2 (5'-GAACCWGCGGARGGATCA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') for the ITS1 region (Schmidt et al., 2013) and the primers ITS86F (5'-GTGAATCATCGAATCTTTGAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') for the ITS2 region (De Beeck et al., 2014). The forward and reverse primers were modified prior to amplification by adding the adaptor sequences 5'-CTTTCCCTACACGACGCTCTTCCGATCT-3' and 5'-GGAGTTCAGACGTGTGCTCTTCCGATCT-3', respectively, which are needed for the multiplexing of PCR products before sequencing. The amplification reaction was performed in a final volume of 25 µl with the modified primers (0.6 µM each), 2 µl of DNA extract, 200 µM of each dNTP, 200 ng/ml BSA, GoTaq® DNA Polymerase (2 units) and 1X Green GoTaq® Reaction Buffer (Promega, Charbonnieres, France), with the following cycling conditions: 94°C for 3 min; 30 cycles of 95°C for 45 s, 52°C (ITS1 region) or 55°C (ITS2 region) for 45 s, 72°C for 45 s; a final elongation step at 72°C for 10 min. To increase richness recovery and to limit PCR bias, three PCR replicates per sample were pooled, evaporated using a vacuum concentrator and resuspended in 50 µl of sterile water. All amplicon products were subjected to paired-end Illumina MiSeq sequencing (2×250 bp) by Get-PlaGe (Genotoul, Castanet-Tolosan, France) as follows, single multiplexing was performed using home-made 6 bp index, which were added to reverse primers during a second PCR with 12 cycles, using the forward primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC) and reverse primer (CAAGCAGAAGACGGCATACGAGAT-index-GTGACTGGAGTTCAGA-CGTGT).The resulting PCR products were purified and loaded onto the Illumina MiSeq cartridge according to the manufacturer instructions. The quality of the run was checked internally using PhiX (20%), and then each pair-end sequences were assigned to its sample with the help of the previously integrated index. Illumina sequencing, base calling and demultiplexing were carried out using RTA v1.18.54, MCS 2.6 and bcl2fastq2.17.