PAX study; early summer collection
Soil cores were collected from 72 forest plots (20x40 m) evenly divided into four treatments (elevated pH, elevated P, elevated pH+P, and untreated control) across 2 physiographic regions of Ohio (one in northern Ohio and one in southern Ohio). Each region contained 36 plots within 3 forest blocks. Within each of the 72 plots, 15 soil cores (2.5 cm diameter, 5 cm depth) were sampled randomly at five time points throughout a calendar year corresponding to distinct stages of tree phenology. A total of 360 soil samples were collected in November of 2010 (Fall), February of 2011 (Winter), April of 2011 for the southern plots and May of 2011 for the northern plots (Spring; sampling was staggered between the regions due to leaf out being later for the northern plots), July of 2011 (Early Summer), and September of 2011 (Late Summer). Soil cores were pooled per plot and sieved to 2 cm. Genomic DNA was extracted from the soils with a standard phenol-chloroform extraction and, for each time point, DNA from the 3 forest blocks was pooled per treatment on an equimolar basis for a final template concentration of 25 ng/?l. This resulted in a total of 120 DNA samples (24 for each time point). The primers ITS1-F and ITS2 were used to PCR-amplify the ITS1 region of the fungal rRNA gene and the primers 27F and 338R were used to PCR-amplify the 16S region of the bacterial rRNA gene in separate reactions. A set of 24 multiplex identifiers were used to separate sequence reads into treatment x region combinations and sequencing was completed on the GS-FLX 454 System with each time point run on a separate 1/8 section of the sequencing plate. Bacterial and fungal PCR products were combined in a 3:1 bacterial:fungal ratio prior to sequencing and separated during demultiplexing.