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Title: Archeorhizomycetes : Nitrogen depletion from litter by Archeorhizomycetes      
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ID:
PRJEB1426
description:
From June to August 2011, soil cores were repeatedly collected at 5 pine forest sites in northern Bavaria, Germany (Table S1). In total, 19 soil cores were collected by using a boring rode (6 cm diam.), from plots with or without Ericacean plants in the understory. The organic layers Oe (partly humified organic layer) and Oa (black, amorphous organic layer) as well as the mineral soil horizons Ah (bleached, dark grey mineral horizon) and E (bleached, pale grey mineral horizon) were separated. The Oe layer was further subdivided into an upper and lower Oe if its thickness exceeded 5 cm. Macroinvertebrates, roots and larger fragments like parts of pine cones were removed and 30 ml of each layer was transferred to two 15 ml tubes for transport. In the laboratory, half of each sample was weighed and dried (60 °C) to constant dry mass in a drying closet for determination of the dry weight. The dried material was subsequently homogenized and used to measure carbon (C) and nitrogen (N) contents in a Multi N/C 2100 analyzer (Analytik Jena, Germany). Based on these data and the thickness of each layer, the changes in C/N ratios per cm depth of the Oe (ΔC/N cm-1) were calculated. Subsamples of 5 g were extracted in 1.0 M KCl solution (soil/solution ratio 1:10), shaken on a horizontal shaker at 225 min-1 for 1h and subsequently filtered (cellulose folded filters 5951/2, 4-7 µm, Whatman, Germany). NH4+ and of NO3- concentrations in the filtered KCL extracts were determined using colorimetric flow injection analysis (FIA-LAB, MLE, Dresden). Activities of enzymes involved in the degradation of the major plant components cellulose (β-glucosidase) and lignin (phenoloxidases) as well as degradation of chitin (chitinase) were comparatively analysed using a 96-well microplate-based system. The activity of the hydrolytic enzymes beta-glucosidase and chitinase were measured using fluorescence photometry subsequent to incubation with 4-methylumbelliferone (MUF) esters (Pritsch et al. 2004). The activity of phenoloxidases was measured using spectrophotometry after incubation with the artificial substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)). To avoid artificial conditions affecting the enzyme activities, the substrate was directly added to the untreated soil without homogenization or addition of buffer solutions. Concentration of total phenolic hydroxyl groups was measured by applying the Folin-Ciocalteu method (Folin & Ciocalteu, 1927), modified according to Bärlocher & Graça (2005). Extracted phenolic compounds were treated with Folin-Ciocalteau reagent and measured with a spectrophotometer (NanoDrop ND-1000 spectrophotometer, Thermo Fisher Scientific Inc., Los Angeles, U.S.A.) at 750 nm. Phenolic content was calculated based on a calibration series with tannic acid (Riedel-de Haën, Seelze, Germany) and expressed as g tannic acid equivalents (TAE) per mg of soil dry mass. Four subsamples of 0.3 g each were weighed from the Oe layer for DNA extraction. DNA was extracted from the soil samples according to Peršoh et al. (2008). Therefor, 300 μl of Al2(SO4)3 (0.4 M) were applied to flocculate humic compounds prior to cell disruption. The fungal ITS1 region was amplified using the Primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA, Gardes and Bruns 1993) and ITS1Univ-R (5’-GCTGCGTTCTTCATCGATGC, White et al. 1990). Primers of each pair were extended by one of 19 different sample identifier sequences (SIDs) of 10 bp in length at the 5’-ends. Each polymerase chain reaction (PCR) batch of 25 μl in total included 15.65 μl H2O, 0.75 μl MgCl2 (50 mM), 2.5 μl 10× PCR buffer (Invitrogen), 2.5 μl dNTPs (2 mM), 1.25 μl of each primer (10 μM), 0.1 μl Taq polymerase (5 U/μl, Invitrogen), and 1 μl DNA extract. Cycling reactions started with an initial denaturation of 2 min at 95°C followed by 35 cycles of 20 s at 94°C (denaturation), 1 min at 61°C (annealing), 2 min at 72°C (elongation) and finished with an extension step of 15 min at 72°C. PCR products were purified using the ExoSAP-IT® kit (Affymetrix) and the relative DNA concentration was assed evaluating images of Agarose gels (0.8%) after electrophoresis (2 µl PCR product and 1 µl of λ-Pst1 DNA ladder) using GeneTools (Syngene). PCR-products were equimolarly pooled and sent on dry ice to GATC (Konstanz, Germany) for 454-pyrosequencing analyses. The 11 525 obtained sequences were assembled to contigs using CLC Genomics Workbench (CLC bio), applying a bubble size of 50 and a minimum contig length of 110. Contigs of at least 90% similarity were initially grouped using the R package ‘RFLP Tools’ R (v2.11.1, www.r-project.org) and revised in BioEdit (v7.1.9, www.mbio.ncsu.edu/BioEdit) by creating consensus sequences of similar sequences (≥ 99% similarity). Groups of which either the 5’-end of the 18S rRNA or the 3’-end of the 5.8S rRNA could not be located were analyzed in detail by downloading and aligning similar sequences. These ambiguous sequences were only retained if they definitely represented non-chimeric ITS1 sequences. Altogether, ITS1 regions were identified and extracted from 187 contigs. These served as reference sequences, against which all 11 525 original sequences were mapped with thresholds for coverage and similarity set to 30% and 95%, respectively. Reference sequences were assigned to operational taxonomic units (OTUs) by grouping full length ITS1 sequences of at least 97% similarity. The abundance of each OTU in each sample was indicated by the number of original sequences successfully mapped to the reference sequences, which applied for 10 812 sequences in total. Names were assigned to OTUs by evaluating BLAST results as detailed previously (Peršoh et al. 2010). Briefly, the GenBank database (www.ncbi.nlm.nih.gov) was searched by BLAST and all hits which attained at least 90% of the best alignment score were considered for name assignment. A taxonomic level unifying all names under which the related sequences were deposited was chosen as name for each OTU. Environmental sequences and clearly outlying names were disregarded, but documented as ambiguities and outliers, respectively. Based on their taxonomic affiliation, OTUs were categorized as ectomycorrhizal (ECM), and ericoid mycorrhizal fungi (ERM). Comparisons with the sequences of Archeorhizomycetes (ARM, Rosling et al. 2011) were conducted separately to identify representatives of this group. To assess the putative biology of the 4 OTUs accounting for more than 3% of all sequences, but neither assignable to ARM, nor representing ECM or ERM fungi, the origin of all best sequences was established. Thereby, OTUs with >95% of the relatives not originating from living organisms were categorized as saprotrophic. This applied to 3 of the 4 OTUs. A matrix coding the abundance of each OTU in each subsample was imported in Primer6 (Plymouth Routines) and abundances within samples were standardized by total. Bray Curtis similarity was applied to calculate similarities among samples. Analyses of Similarity (ANOSIM) were used to elucidate if the understory vegetation had an impact on composition of fungal community as a whole, on the ARM community, and on the ECM, ERM, and saprobic communities. Similarities among samples were visualized by non-metric multidimensional scaling. Correlations among distribution patterns of OTUs, C/N ratios and contents, N fractions, and enzyme activities were assessed by calculating Pearson Product Moment Correlations of Log10 + 1 transformed data using the software R version 2.13.1. References Bärlocher, F, Graça, MAS (2005). Total phenolics. In: Graça, MAS , Bärlocher, F , Gessner, MO (eds.). Methods to study litter decomposition - A practical guide. Springer pp 97–100. Folin, O, Ciocalteu, V (1927). On tyrosine and tryptophane determination in proteins. J Biol Chem 27, 239–343. Gardes M, Bruns TD (1993). ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol Ecol 2: 113–118. Peršoh D, Theuerl S, Buscot F, Rambold G (2008). Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil. J Microbiol Methods 75: 19–24. Peršoh D, Melcher M, Flessa F, Rambold G (2010). First fungal community analyses of endophytic ascomycetes associated with Viscum album ssp. austriacum and its host Pinus sylvestris. Fungal Biol 114: 585–596. Pritsch K, Raidl S, Marksteiner E, Blaschke H, Agerer R, Schloter M, Hartmann A (2004). A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system. J Microbiol Methods 58, 233–241. Rosling A, Cox F, Cruz-Martinez K, Ihrmark K, Grelet G-A, Lindahl BD et al (2011). Archaeorhizomycetes: Unearthing an Ancient Class of Ubiquitous Soil Fungi. Science 333: 876–879. White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Shinsky JJ, White TJ (eds). PCR Protocols: A Guide to Methods and Applications. Academic Press. pp 315–322.
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJEB1426
authentication:
none
authorization:
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dateReleased:
02-12-2013
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject

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