Plant-microbe relationships in restored and natural tidal freshwater wetlands
Samples were collected from three restored and two natural reference TFWs: One natural (Jug Bay, N38.78580 W76.71308) and one restored (Wootons Landing, N38.85646 W76.69124) site were located in the suburban area of central Maryland on the Patuxent subestuary of Chesapeake Bay. In the Anacostia watershed, a natural remnant wetland (Dueling Creek, N38.92411 W76.94018) was selected along with two restored marshes, one restored in 1992-93 (Kenilworth, N38.91035 W76.94588) a second in 2000 (Kingman, N38.90414 W76.96182). For each of the sites, three replicated stands of four common plant species were targeted: Typha latifolia L., Peltandra virginica (L.) Schott, Lythrum salicaria L., and Phragmites australis (Cav.) Trin. ex Steud. ssp. australis. Each site contained areas dominated by these four species, with the exception of Lythrum, which was absent in Wootons. This study design resulted in a total of 57 collected samples. A half circular Russian peat borer was used to collect two 5.2 x 50-cm cores. In each plot, soils were sampled <1 cm away from the clipped shoots of the species of interest. For each sample plot, two soil cores were collected and homogenized into a single representative sample. Soil samples were thoroughly mixed, and ~10 g of soil was removed from each sample and stored at -20°C until DNA extraction.      Pyrosequencing was used to investigate microbial community structure.  PCR reactions were set up using Promega GoTaq® DNA Polymerase (Promega Corporation, Madison, WI) following the protocol described by Bates, et al. 2011. Each reaction was set up using primers F515 (5’-GTGCCAGCMGCCGCGGTAA-3’) and R806 (5’-GGACTACVSGGGTATCTAAT-3’) targeting a 291 bp fragment in the V4 and V5 region of 16S rRNA genes.  Multiplexing and sequencing of all 57 samples was accomplished using a 10-bp MIDS barcoded F515 primer also containing a Roche 454-A pyrosequencing adaptor (5’- CCATCTCATCCCTGCGTGTCTCCGACTCAG-3’; Roche Applied Science, Branford, CT, USA) and a “GA” linker sequence. Target sequences were amplified in a 25 µl PCR reaction.  Each reaction contained 0.20 µM forward and reverse primers, 0.20 mM of dNTPs, 1.75 mM MgCl2, 1X GoTaq® Colorless Flexi Buffer (Promega Corporation, Madison, WI) with 1.5 mM MgCl2, 0.064% BSA, and 0.025 Taq U/µl GoTaq® Hot Start Polymerase (Promega Corporation, Madison, WI).  PCR reaction conditions began with a 95°C heat activation step for 5 min followed by 30 cycles of 95°C for 15 sec, 55°C for 30 sec, and 72°C for 30 sec, with a final extension step at 72°C for 60 sec.  Post-amplification, each barcoded PCR product was purified following the UltraClean PCR Clean-Up Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA) except 4.5X SpinBind solution was mixed with the 25 µl product.  Samples were sent to the Institute for Genome Sciences and Policy (Duke University, Durham, NC) and analyzed using titanium chemistry on a Roche 454 GS-FLX (Roche Applied Sciences, Penzberg, Germany).
University of Maryland
MAES_WETLANDS_PRASSE