Maize crop with synthetic plastic (LDPE) mulch film
16S rRNA gene amplicon sequencing was conducted for each treatment (n = 3-4). At the end of the experiment, three soil samples (0-20 cm depth) per plot were taken randomly within a 15 cm radius around the plants and homogenised (total ca. 50 g). Samples were stored in 50 ml sterile centrifuge tubes and immediately frozen at -80 C before freeze-drying. Bacterial DNA was extracted using the Zymo Research Quick DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA) according to manufacturers instructions. Quality and concentration of extracted DNA were assessed by gel electrophoresis and by Qubit 4.0 Fluorometer dsDNA BR Assay Kit (Life Technologies, Carlsbad, CA). Libraries of 16S rRNA gene amplicons were prepared by duplicate PCR with double-indexed fusion primers as described in Fadrosh et al. (2014). The hypervariable V4 16S rRNA gene fragment was amplified using modified forward primer F515 (5-GTGBCAGCMGCCGCGGTAA-3) and reverse R806 prokaryotic primer (5-GGACTACHVGGGTWTCTAAT-3), which amplify an approximately 290 bp region. Primers were designed to contain: the Illumina adapters and sequencing primers, a 12 bp barcode sequence, a heterogeneity spacer to mitigate the low sequence diversity amplicon issue, and 16S rRNA gene universal primers (Fadrosh et al., 2014). PCRs were performed using OneTaq DNA Polymerase (New England Biolabs, Ipswich, MA, USA). All reactions were run with no-template negative controls. Thermocycling conditions were as follows: initial denaturation at 95C for 2 min, followed by 30 cycles at 95C for 30 s, 50C for 50 s, and 68C for 90 s with a final elongation at 68C for 5 min. Amplicons were visualised in a 1.2% tris-acetate agarose gel using a GelDoc System (Bio-Rad, Hercules, CA). DNA bands of approximately 440 bp were gel-purified using the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). The purified amplicons were then quantified using Qubit 4.0 Fluorometer, pooled in equimolar amounts and the final pool was run on Illumina MiSeq platform (Illumina Inc., San Diego, CA) using 500-cycle v2 chemistry (2  250 bp paired-end reads) at the Centre for Environmental Biotechnology, Bangor, United Kingdom.