60 of Turnquist et al.: An Examination of Soil Microbial Communities and Litter Decomposition five randomly selected sampling areas and homogenized into one representative soil sam- ple. Approximately half of the homogenized soil samples were placed in paper bags, air dried, passed through a 2 mm sieve, and stored at room temperature until physical and chemical analysis. The remaining soil was immediately placed on ice, frozen within 12 hours, and stored at -20°C prior to biological analysis. Physical and Chemical Soil Analysis Physical characteristics measured for each site included: percent sand, silt, and clay measured by the hydrometer method (Gee and Bauder 1986); soil organic matter (SOM) content (per- cent loss on ignition, Kalra and Maynard 1991); percent volumetric water content at field capac- ity (pressure plate technique at one third bar, Gardner 1986); and bulk density as measured by averaging two 68.7 cm3 oven-dried soil samples (Blake and Hartage 1986) independently col- lected with an ICT International model 0200 Soil Bulk Density Sampler (ICT International, Australia) at two randomly selected undisturbed locations in each study site. Chemical analyses consisted of total carbon (C), and total nitro- gen (N) as measured on a CE 2000 Carbon ni- trogen analyzer (Carlo Erba), 2:1 water:soil pH (electrode), extractable phosphorus (Bray), am- monium acetate extracted calcium (Ca), mag- nesium (Mg), potassium (K), sodium (Na), and cation exchange capacity (CEC), by sum- mation of the base cations Ca, Mg, K, and Na. Molecular Community Fingerprint Analysis with TRFLP Terminal restriction fragment length poly- morphism (TRFLP) was used to profile the bacterial and fungal communities (Liu et al. 1997). All molecular analyses were per- formed in the University of Wisconsin–Stevens Point Molecular Conservation Genetics Lab- oratory (MCGL). Microbial DNA was ex- tracted from 0.25 g of soil using a variation of the modified Burgmann method (Thakuria et al. 2008). The variation consisted of re- placing the final polyvinylpolypyrrolidone spin column with a modified gel purifica- tion method according to Zhang et al. (2009). ©2016 International Society of Arboriculture Reaction mixtures for bacterial 16S rDNA PCR amplification contained 1 µl template DNA, 1× Taq buffer (New England Biolabs, Ipswich, Massachusetts, U.S.), 600 µM dNTPs, 3.0 mM MgCl2 , 0.25 mg bovine serum albumin (BSA), primer, and 2.5 U Taq DNA polymerase (New England Biolabs) in a final volume of 50 µl. The primers were PET labeled fun18S1 (PET- CCATGCATGTTAAGTWTAA) and fung5 (GTAAAAGTCCTGGTTCCCC) (Anderson and Cairney 2004). Fungal PCR conditions con- sisted of a 5-min hot start at 95°C, followed by 35 cycles of 95°C/30 sec, 53°C/30 sec, 72°C/1 min with a final extension of 72°C/15 min. Three PCRs of each sample were performed on a GeneAmp 9700 (Applied Biosystems, Foster City, California, U.S.), and the result- ing products were pooled to minimize bias. The pooled products were cleaned and con- centrated using a DNA Clean & Concentrator- 25 Kit (Zymo Research, Irvine, California, U.S.) following the manufacturer’s protocol. Multiple single digests of the concentrated bacterial and fungal PCR products were com- pleted using 10 U of restriction enzyme for two hours at 37°C. The bacterial products were digested with HhaI, MspI, and RsaI (New Eng- land Biolabs) and renamed B1, B2, and B3, respectively. Similarly, fungal PCR products were digested with AluI, MspI, and RsaI (New England Biolabs) and renamed F1, F2, and F3, respec- tively. Terminal restriction fragments (T-RF) were visualized using a 3730xl Genetic Analyzer (Applied Biosystems) with LIZ™ 1200 internal size standard (Applied Biosystems). Fragments were sized using Genemapper v4.0 analysis 150 µM each primer, and 2.5 U Taq DNA poly- merase (New England Biolabs) in a final volume of 50 µl. The primers were 6-FAM labeled 8f (6FAM-AGAGTTTGATCCTGGCTCAG) and 926r (CCGTCAAATCCTTTRAGTTT) (Hackl et al. 2004). Bacterial PCR conditions con- sisted of a 5-min hot start at 95°C, followed by 25 cycles of 95°C/1 min, 53°C/1 min, 72°C/1 min with a final extension of 72°C/15 min. For fungal 18s rDNA amplification, the PCR reac- tion contained 1 µl template DNA, 1 × Taq buffer (New England Biolabs), 600 µM dNTPs, 2.0 mM MgCl2 , 0.25 mg BSA, 100 µM each
January 2016
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