Ecological Archives E096-288-A1

Lauren C. Cline and Donald R. Zak. 2015. Soil microbial communities are shaped by plant-driven changes in resource availability during secondary succession. Ecology 96:33743385. http://dx.doi.org/10.1890/15-0184.1

Appendix A. Supplemental information used to quantify root biochemistry, extracellular enzyme activity, and functional gene abundance, as well as amplify fungal and bacterial DNA.

Environmental Characteristics

Root lignin was determined by the acid detergent lignin (ADL) procedure, in which ADL is determined gravimetrically as the residue remaining upon ignition after H2SO4 treatment (Goering and Van Soest 1970). Root cellulose was calculated by subtracting percent acid detergent fiber (ADF) and lignin from root dry mass. ADF was determined gravimetrically as the residue remaining after dissolution and extraction of cell solubles, hemicellulose and soluble minerals with hexadecyltrimethylammonium bromide and sulfuric acid. Hemicellulose was determined by subtracting ADF and neutral detergent fiber (NDF; Van Soest et al. 1991).

Microbial Community Analysis

Triplicate 28S PCR reactions for each composite soil sample contained 1 µL of DNA, 0.5 µL of 20 µM forward primer, 0.5 µL of 20 µM reverse primer, 2.5 µL dNTPs (2 µM), 2.5 µL 10X PCR buffer (1.5 mM MgCl2; Roche, Hamburg, Germany), 1 µL BSA, 0.5 µL high-fidelity Taq polymerase (Roche, Hamburg, Germany), and 16.5 µL molecular grade water. Following an initial denaturation step at 95 °C for 10 min, fungal PCR was cycled 34 times at 95 °C for 1 min, 54 °C annealing temperature for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 7 min. Negative controls were included in every PCR reaction. Triplicate 16S PCR reactions contained 1 µL of DNA, 0.5 µL of 20 µM forward primer 27f, 0.5 µL of 20 µM reverse primer 519r, 0.5 µL dNTPs (20 mM), 5.0 µL 10X PCR buffer (1.5 mM MgCl2; Roche, Hamburg, Germany), 0.6 µL high-fidelity Taq polymerase (Roche, Hamburg, Germany), and 16.9µL molecular grade water. Following an initial denaturation step at 95 °C for 10 min, PCR was cycled 26 times at 94 °C for 30 s; we used a 55 °C annealing temperature for 1 min, 72 °C for 90s, and a final extension at 72 °C for 20 min.

Briefly, sequences were sorted by barcode and trimmed, removing primers and barcodes, followed by alignment to 16S and 28S reference alignments (James et al. 2006, Quast et al. 2013). DNA contaminants (chloroplast, mitochondrial and unknown DNA) and chimeras, identified using uchime (Edgar et al. 2011), were removed from downstream analysis.

Extracellular Enzyme Analysis

One gram of soil mixed and homogenized in 125 mL of 50 mM sodium acetate buffer for 1 minute. Enzyme assays included 100 mL soil-buffer solution, in addition to 50 mL of substrate. Controls included soil plus buffer, buffer plus substrate, and, for MUB substrates, soil and MUB. Cellobiohydrolase, b-xylosidase, b-1,4-glucosidase and N-acetyl-b-glucosaminidase assays were incubated in the dark at 20 °C for 2 h or 30 minutes. Afterwards, 25 mL of 0.2 M NaOH was added to each well to stop the reaction and increase fluorescence. Enzyme activity was measured in a Molecular Devices f-MAX fluorometer set at 365 nm excitation wavelength and 460 nm emission wavelength. Phenol oxidase and peroxidase assays were incubated for 24 h and rates were estimated spectrophotometrically (Saiya-Cork et al. 2002).

Table A1. Summary of functional genes used in metagenomic analysis of bacterial functional potential to degrade plant and microbial litter.

Enzyme (Gene) Name

EC Number

Substrate Category

Beta-glucosidase

3.2.1.21

Cellulose

Endocellulase

3.2.1.4

Cellulose

Alpha-N-acetylglucosaminidase

3.2.1.50

Chitin

Chitin deacetylase

3.5.1.41

Chitin

Endochitinase

3.2.1.14

Chitin

β-N-acetylhexosaminidase

3.2.1.52

Chitin

Alpha galactosidase

3.2.1.22

Galactose-containing Oligosaccharides

Laccase

1.10.3.2

Lignin

Endopolygalacturonase

3.2.1.15

Pectin

Exopolygalacturonase / galacturan 1,4-α-galacturonidase

3.2.1.67

Pectin

Pectate lyase

4.2.2.2

Pectin

Pectin lyase

4.2.2.10

Pectin

Pectinesterase

3.1.1.11

Pectin

Alpha-amylase

3.2.1.1

Starch

Alpha-glucosidase

3.2.1.20

Starch

Glucan 1,4-alpha-glucosidase

3.2.1.3

Starch

Alpha-L-arabinofuranosidase

3.2.1.55

Xylan

Beta-xylosidase

3.2.1.37

Xylan

Endo-1,4-ß-xylanases

3.2.1.8

Xylan

 

Table A2. Summary of functional genes used in metagenomic analysis of fungal functional potential to degrade plant litter.

Enzyme (Gene) Name

EC Number

Substrate Category

Cellulose 1,4-beta-cellobiosidase (cbh1)

 3.2.1.91

Cellulose

B-glucosidase

3.2.1.21

Cellulose

α-N-acetylglucosaminidase

3.2.1.50

Chitin

β-N-acetylhexosaminidase

3.2.1.52

Chitin

Alpha galactosidase

3.2.1.22

Galactose-containing Oligosaccharides

Laccase (lcc)

1.10.3.2

Lignin

Manganese peroxidase (mnp)*

1.11.1.13

Lignin

Lignin peroxidase (lip)*

1.11.1.14

Lignin

Versatile peroxidase (vp)*

1.11.1.16

Lignin

Endopolygalacturonase

3.2.1.15

Pectin

Beta-xylosidase

3.2.1.37

Xylan

Alpha-L-arabinofuranosidase

3.2.1.55

Xylan

* Due to similarities in functional genes and relatively short reads from Illumina sequencing, a single gene database was created.

Literature cited

Edgar, R. C., B. J. Haas, J. C. Clemente, C. Quince, and R. Knight. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–200.

Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analysis. USDA Agricultural Research Service Handbook number 379. U.S. Dept. of Agriculture: Washington, D.C., USA.

James, ,T. Y., F. Kauff, C. L. Schoch, P. B. Matheny, V. Hofstetter, C. J. Cox, et al. 2006. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature 443:818–22.

Quast C., E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza, et al. 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–6.

Saiya-Cork, K., R. Sinsabaugh, and D. Zak. 2002. The effects of long term nitrogen deposition on extracellular enzyme activity in an Acer saccharum forest soil. Soil Biol Biochem 34:1309–1315.

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. Journal of Dairy Science 74:3583–3597.


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