title: ITS-Fungi
design: Soil DNA was extracted from 0.25 g soil using the PowerSoil DNA isolation kit (MoBio, Carlsbad, CA) following the instructions of the manufacturer. DNA concentrations were determined by NanoDrop spectrophotometry (NanoDrop, Wilminghton, DE). The abundance of bacteria and fungi was assessed by quantitative PCR using the Qiagen QuantiTect SYBR Green PCR Master Mix (Qiagen Inc., Victoria, Australia), a Cavro Omni Robot (Tecan Group Ltd., Seestrasse, Mannedorf, Switzerland) and an ABI 7900 real-time PCR machine (Applied Biosystems, Foster City, CA). Bacterial 16S rRNA was quantified using 338F and 518R primers (Lane et al., 1985; Muyzer et al., 1993) and fungal ITS was quantified using ITS 1F and ITS4 primers (Gardes and Bruns, 1993; White et al., 1990). Sequencing and bioinformatics analyses were carried out according to the Biomes of Australian Soil Environments (BASE) protocol (Bissett et al., 2016). Amplicons targeting the bacterial 16S rRNA gene were prepared using the 27F (AGAGTTTGATCMTGGCTCAG) and 519R (GWATTACCGCGGCKGCTG) (Lane et al., 1985; Muyzer et al., 1993). Fungal ITS gene regions were targeted using ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS4 (TCCTCCGCTTATTGATATGC) primers (Gardes and Bruns, 1993; White et al., 1990). These primer pairs were selected based on the BASE project where they were found suitable for assessing bacterial and fungal communities (Bissett et al., 2016). For all amplicons, Illumina 300 bp paired-end sequencing was performed at the Australian Genome Research Facility (Melbourne, Australia).