Soil fungi in a temperate hardwood forest profiled over five years.

Soil cores were collected monthly from six long-term monitoring sites within an 80-hectare old growth (>200 years old) temperate forest located in northeastern Ohio, USA over five years (November 2006 through October 2011). Using terminal restriction fragment length polymorphism (TRFLP) on 997 soil samples collected at three soil depths over the five years, it was found that soil fungi responded strongly to soil phosphorus availability and soil moisture. Thus, 96 of the 997 soil samples at the 2-cm depth only were selected for high throughput sequencing that represented low or high moisture conditions (<27% or >37% gravimetric soil moisture respectively) and high or low levels of available phosphorus (mean total bicarbonate available P 196 ± 11 µg/g and 78 ± 9 µg/g respectively). Amplicons of the fungal ITS-2 region were made with the primers 58A2F and NLB4 containing Illumina overhang adapters. Amplicons were purified, indexed, and sequenced as 2 x 250bp reads on one lane of the Illumina MiSeq V3 sequencer at the Case Western Reserve University Genomics Core.