Microbiome of bulk soil, rhizosphere, roots and nodules of Pisum sativum Raw sequence reads

We studied how different P fertilizers affect NRE communities in nodules. The field trial was conducted in Braunschweig, Germany. Dystric Cambisols and Orthic Luvicols were the predominant soil types developed in silty sandy soil. This experiment used a randomized block design with three replicates. A five-year crop rotation was adopted, including winter barley, winter oilseed rape, winter wheat, lupin, and winter rye. Deep chisel plowing and conventional plowing to a depth of 25 cm were employed to incorporate the remaining straw and stubble before sowing. Four fertilizer treatments with increasing solubility were tested: bone char (BC), and bone char plus (BCplus) as alternative fertilizer, and compared it to triple superphosphate (TSP) and no P fertilizer (P0). Samples from the bulk soil, rhizosphere, roots, and nodules were taken during the late flowering stage (BBCH 68/69) of Pisum sativum with the cultivar Salamanca. Late flowering as this is the stage where nodule biomass peaks. The bulk soil samples are composed of 15 punches (0-10 cm) per plot. To increase the sample material, two closely spaced plants were pooled together for the rhizosphere, root, and nodule samples. The rhizosphere soil was carefully removed from the root of the two plants per plot. Bulk soil and rhizosphere samples were sieved at 2 mm. The nodules were removed from the roots, and both were subsequently separately surface sterilized in the field with sodium hypochlorite. A 30-cycle-long PCR was performed with the remaining washing water, and a surface sterile sample was incubated overnight on agar plates to ensure that the surface sterilization was successful. We applied DNA extraction and 16S rRNA gene metabarcoding using the primers of the earth microbiome project (EMP, 515F/806R , Parada et al., 2016, Apprill et al., 2015) . Amplification: 98C for 1 min; 98C for 10 s / 55C for 30 s / 72C for 30 s (23 cycles); 72C for 5 min. PCR reaction mixture (total 25 ul); 12.5 ul of NEB Next High Fidelity Master Mix, 0.3 ul of each primer(10 pmol), 2.5 ul of 3% BSA, 1 ul of template DNA (10 ng/ul), an adequate amont of DPEC-treated water. Sample purification: purified with MagSi-NGS Prep Plus magnetic beads (Steinbrenner Laborsysteme GmbH, Mannheim, Germany) using a beads: DNA ratio of 0.8:1. Indexing PCR: Nextera XT Index Kit v2, Amplification: 98C for 30s; 98C for 10 s / 55C for 30 s / 72C for 30 s (8 cycles); 72C for 5 min. Sample purification: mentioned above. Products are pooled equimolar to a final concentration of 4 nM and sequenced using the MiSeq Reagent kit v3 (600 cycles) for paired end sequencing. Raw sequences were separated from adapters using AdapterRemoval v2.1.7, and reads were trimmed with a minimum read length of 20 bp.