DOI: 10.25829/bexis.31402-8
title: Frankia sp. is governing the diazotrophic community composition in deadwood of 13 tree species (diazotrophic community in the total fraction)

description: Deadwood is a nitrogen (N) poor environment and its decomposition rate is limited by N-bioavailability. Diazotrophs can reduce such N-limitations as they are capable to fix N2, which employ the functional gene nifH that encode for dinitrogenase reductase. The present study investigates the diazotrophic community composition and activity, and their relationship to environmental factors and wood parameters of 13 different tree species stored as logs in temperate forests. Directly after sampling, each deadwood sample was incubated with water (total fraction) or bromodeoxyuridine (BrdU), which labeled the metabolically active fraction. Thereafter, nucleic acids extract from both total and active fraction were used to assess the nifH gene-based diversity by amplicon sequencing.Here, the dataset contain the diazotrophic community of the total fraction in deadwood logs of13 tree species in 9 temperate forest plots (Hainich-Dün exploratory).<br>The data collection and analysis was part of the project Woodstock (subproject of the BELongDead project) within the framework of the DFG-funded joint project Biodiversity Exploratories (https://www.biodiversity-exploratories.de).
description: Detailed description of study design: In winter 2008/09, freshly cut logs of 13 different tree species were placed on the forest soil in random order and within 1m distance to each other. The logs were 4m long and around 31cm in diameter. Various parameters have been measured throughout the years. For further details on the study design see: Kahl et al. 2017.<br>In June 2020, after 11 years of decomposition, deadwood samples of the outer 7 cm of the BELongDead logs in the Hainich-Dün exploratory were taken on 9 experimental plots (EP). Bark and leaves were removed. The samples were further processed in the laboratory.<br>Sampling design: The outer 7 cm of the logs were cut laterally from the stem with an electric rip saw and chiseled out (about 2cm wide). For each of the 13 tree species per plot, one sample was taken.<br>Sample preparation: In June 2020, deadwood samples were cut laterally with an electric saw from the outer 7 cm of each log. Deadwood samples consisted almost exclusively of sapwood, but mixtures of sapwood and heartwood were taken from Quercus logs due to the decay and loss of sapwood. One part of the sample was stored in a plastic bag in the field and kept cool (∼5 °C) until further treatment. A second part of the sample (sawdust) was transferred into a 15 mL falcon tube in the field. After that, 10 mM bromodeoxyuridine (BrdU) was added sufficiently until the sawdust was completely soaked with solution. The falcon tubes were placed in an upright position and covered with aluminum foil, followed by incubation at room temperature for 48 h. The samples were then frozen at −80 °C in the laboratory before the characterization of the diversity of metabolically active diazotrophs.brSample analysis: DNA was isolated from the BrdU-treated deadwood samples using Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo, California, USA) according to the manufacturer’s instructions. The extracted DNA included all types of genomic DNA (dormant cells, dead cells, metabolically active, and replicating cells), we named total fraction of deadwood samples. BrdU immunocapture approach (McMahon et al. 2011) was employed to separate the BrdU-labeled DNA from the total fraction . Briefly, for each sample, 2 μL monoclonal BrdU antibodies (1 mg μL–1 mouse anti-BrdU, clone BU-33, Sigma-Aldrich) was mixed with18 mL denatured herring sperm DNA (1.25 mg mL–1 in phosphate buffer saline (PBS), Promega, Walldorf, Germany) and then incubated 45 min at 30°C to form antibody-herring sperm DNA complex. Denatured sample DNA (25 μL; ∼200 ng of extracted DNA + 10 μL PBS) was then added to antibody-herring sperm DNA complex and incubated for 30 min at 30°C to capture BrdU-labeled DNA. Thereafter, the mixture was added to 6.26 μL aliquots of washed DynabeadsTM goat anti-mouse IgG (Invitrogen) in PBS–bovine serum albumin solution (PBS–BSA) and gently rotated for 30 min at room temperature on an RM-2 roll mixer (Carl Roth GmbH, Karlsruhe, Germany) to form the Dynabead complex (Dynabead-BrdU antibodies-BrdU-labeled DNA). The Dynabead complex was washed with 100 mL PBS–BSA eight times by adding the wash solution, and trapping the complex with a magnetic particle concentrator (Dynal). BrdU-labeled DNA was isolated from the washed Dynabeads by adding 20 μL BrdU solution (1.7mM in PBS–BSA) followed by 35 min incubation under slow rotation. The obtained BrdU-labeled DNA was thereafter named as active fraction.brEquipment: CFX96™ Real-Time System (Bio-Rad, Feldkirchen, Germany),Petroff counting chamber (Paul Marienfeld GmbH, Germany),Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo, California, USA) and SYBR® Green Supermix (Bio-Rad, Feldkirchen, Germany)br Data analysis: Illumina generated demultiplexed paired-end sequences were merged using the BBMerge v34.48 (Bushnellet al. 2017). The obtained combined sequences were initially undergone for quality filtering employing DADA2 algorithm , Version1.24.0 (Callahan et al. 2016) that resolves amplicon-sequencing errors to generate amplicon sequence variants (ASVs). Non-target-length sequences, which could potentially be introduced as a result of non-specific priming, were removed from the sequence table. Only sequences with a length between 250 and 400 bp were kept.brLiterature references: Kahl T, Arnstadt T, Baber K (2017) Wood decay rates of 13 temperate tree species in relation to wood properties, enzyme activities and organismic diversities. For Ecol Manag 391:86–95. https://doi.org/10.1016/j.foreco.2017.02.012brCallahan BJ, McMurdie PJ, Rosen MJ (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583. https://doi.org/10.1038/nmeth.3869brMcMahon S, Wallenstein M, Schimel J (2011) A cross-seasonal comparison of active and total bacterial community composition in Arctic tundra soil using bromodeoxyuridine labeling. Soil Biol Biochem 43:287–295. https://doi.org/10.1016/j.soilbio.2010.10.013brBushnell B, Rood J, Singer E (2017) BBMerge – Accurate paired shotgun read merging via overlap. PLOS ONE 12:0185056. https://doi.org/10.1371/journal.pone.01850brData references: DADA2 (https://benjjneb.github.io/dada2/tutorial_1_2.html, Version1.24.0)brBBMerge (https://sourceforge.net/projects/bbmap/)